Hugo Sovino

Clomiphene citrate and ovulation induction.

Clomiphene can be used to treat anovulation due to hypothalamus or pituitary gland dysfunction, and it normalizes the luteal phase in stimulated patients. It can be used to estimate ovarian follicle reserve, and may be predictive of ovulation in women aged >/=35 years or with failed IVF. Contraindications include risk of congenital anomalies, chronic liver disease and visual disorders. Clomiphene may impair fertility through its effects on cervical mucus and in causing various endometrial dysfunctions. However, if clomiphene is administered in 50 mg doses, side-effects are avoided and efficacy is similar to that of a 100 mg dose, although daily dosages of 200 mg/day over 5 days can induce ovulation in approximately 70% of treated patients. Gonadotrophin concentrations increase up to days 5-9 when follicles are selected, and clomiphene is effective in patients with polycystic ovary syndrome (PCOS). Fifty percent of normal patients conceive, a value perhaps biased by the antagonistic effects of clomiphene on cervical mucus in some women. Clomiphene is valuable for IVF, and is used by some clinics in combination with HMG or recombinant FSH. Resistance to clomiphene can develop, and human chorionic gonadotrophin may be needed to induce ovulation in clomiphene cycles. Corticosteroids and human menopausal gonadotrophin (HMG) can be combined with clomiphene for stimulation, its combination with HMG long having been a standard protocol in assisted reproduction. PCOS patients may become insulin resistant, a condition improved by the administration of metformin. Other adverse effects include multiple pregnancies, an increase in the rate of multiple births, ovarian hyperstimulation and unsubstantiated claims of ovarian cancer.

Nuclear factor κB pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis

In order to investigate the role of the nuclear factor kappaB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IkappaB kinase (CHUK), NFKBIA and IL6 expressions and NFKB DNA binding in eutopic endometrium from women with endometriosis. Eutopic endometrium was obtained from 37 women with endometriosis and 42 fertile women during laparoscopy. We analysed RELA, CHUK, NFKBIA and IL6 mRNA levels (RT-PCR); RELA, CHUK and NFKBIA proteins and p-NFKBIA/NFKBIA ratio (western blot); and NFKB binding (DNA shift assay) and IL6 concentration (ELISA) in endometrial explants. Our results indicate that mRNA and cytoplasmic proteins of RELA and CHUK exhibit constant levels in normal endometrium during the menstrual cycle. A dramatic increase (P<0.05) in NFKBIA mRNA expression, RELA nuclear presence and the mRNA and the protein of IL6 during late secretory phase was also observed in this tissue. By contrast, in eutopic endometrium from endometriosis patients, a decrease (P<0.05) in IL6 mRNA and protein (61%), NFKBIA mRNA (46%), p-NFKBIA/NFKBIA ratio (42%), RELA nuclear stromal (68%) and CHUK (48%) proteins were found exclusively during the late secretory phase compared with normal endometrium. In conclusion, the canonical activation of NFKB pathway is deregulated and may have reduced transcriptional function affecting NFKBIA and IL6 expression, genes related local proinflammatory processes. These molecular alterations observed during the late secretory phase in eutopic endometrium from endometriosis patients constitute a NFKB system dysfunction, suggesting that NFKB could be an important factor in endometriosis aetiology.

Physiologic activation of nuclear factor kappa-B in the endometrium during the menstrual cycle is altered in endometriosis patients.

OBJECTIVE To evaluate nuclear factor kappaB (NF-κB) activation and NF-κB-p65 subunit activation, immunolocalization, and expression in the endometrium of healthy women and endometriosis patients throughout the menstrual cycle. DESIGN Prospective observational study. SETTING Affiliated hospital and university research laboratory. PATIENT(S) Twenty-four healthy women and 24 endometriosis patients. INTERVENTION(S) Menstrual, proliferative, and secretory endometrial biopsies. MAIN OUTCOME MEASURE(S) Assessment of NF-κB and p65 activation by protein-DNA binding assays and p65 localization and expression by immunohistochemistry. RESULT(S) Total NF-κB-DNA binding was constitutive and variable in human endometrium accross the menstrual cycle. Healthy women (physiologic conditions) showed higher p65-DNA binding in proliferative than in menstrual and secretory endometrium. Conversely, in endometriosis patients, p65-DNA binding was higher in proliferative and secretory endometrium than in menstrual endometrium. Endometrial epithelial cells showed higher p65 expression level score than endometrial stromal cells. CONCLUSION(S) NF-κB activity is constitutive, physiologic, and variable in human endometrium. The physiologic cyclic p65 activation pattern was altered in endometriosis patients, showing no cyclic variation between the proliferative and secretory phase of the menstrual cycle. The absence of decreased p65 activity in secretory endometrium from endometriosis patients is concurrent with progesterone resistance and could participate in endometrial biologic alterations during the implantation window in endometriosis patients.

Association between MMP1 and MMP9 activities and ICAM1 cleavage induced by tumor necrosis factor in stromal cell cultures from eutopic endometria of women with endometriosis

Endometriosis is a benign gynecological pathology in which immune system deregulation may play a role in its initiation and progression. In endometriotic lesions, intercellular adhesion molecule-1 (ICAM1) is released from the cell membrane by proteolytic cleavage of its extracellular domain, a process that coincides with increased expression and proteolytic activity of metalloproteinases such as MMP1 and MMP9. The objective of our study was to investigate the association between MMP1 and MMP9 activities and ICAM1 cleavage mediated by tumor necrosis factor (TNF) in eutopic endometrial stromal cells from women with and without (control) endometriosis during culture. The RNA was evaluated by RT-PCR, and the protein was determined by western blot (ICAM1, MMP1), casein or gelatin zymographies (secreted active MMP1 or MMP9 respectively), ELISA (soluble ICAM1 (sICAM1)), and fluorescence assay (secreted active MMP1). Under basal conditions, proMMP9 dimer and MMP9 were higher in endometriosis cell cultures. In stromal cultures derived from control women and those with endometriosis, TNF augmented the intracellular proMMP1 (1.2-fold in control stromal cells) and ICAM1 (1.4- and 1.9-fold), greatly increased MMP1 and proMMP9 levels, and the sICAM1 concentration (2.3- and 4.3-fold) in their media compared with basal levels. The combination of TNF and MMP9 increased the sICAM1 concentration 14-fold in the endometriosis cell media, whereas GM6001 inhibited the stimulatory effect of TNF in both cell cultures. The deregulation of MMP9, and the TNF participation in the MMP1 and proMMP9 secretions, in the MMP9 expression and in the expression and cleavage of ICAM1 may contribute to the pathophysiology of this disease.

P450Arom induction in isolated control endometrial cells by peritoneal fluid from women with endometriosis

OBJECTIVE To study the effect of peritoneal fluid from women with (PF-E) and without (PF-C) endometriosis on P(450)Arom expression in endometrial cells. DESIGN Experimental study. SETTING University research unit. PATIENT(S) Forty women of reproductive age with (n = 22) or without (control; n = 18) endometriosis. INTERVENTION(S) Peritoneal fluid and eutopic endometrial samples were obtained during surgery from women with (n = 13 and 9, respectively) and without (n = 4 and 14, respectively) endometriosis. MAIN OUTCOME MEASURE(S) Expression study for P(450)Arom, steroid factor 1 (SF-1), chicken ovalbumin upstream transcription factor I (COUP-TFI), and COUP-TFII messenger RNA (reverse transcriptase-polymerase chain reacion) and/or protein (immunoblot) in isolated endometrial epithelial cells transfected or not with expression vector containing SF-1, COUP-TFI, or COUP-TFII complementary DNAs. RESULT(S) Basal messenger RNA and/or protein expression of P(450)Arom and SF-1 were augmented in endometriosis, and that of COUP-TF was diminished. In control cells, (Bu)(2)cAMP and PF-E increased P(450)Arom and SF-1 expression (but not COUP-TF expression) in a dose-dependent way, an effect not observed with PF-C, adsorbed PF-E, or 10(-5) M indomethacin. Transfected cells confirmed these results. Any treatments modified the studied molecules in endometriosis cells. CONCLUSION(S) These data indicate that molecules contained in PF-E favor an estrogenic microenvironment, suggesting a role in the etiopathogenesis of endometriosis enabling the survival, maintenance, and growth of endometrial implants in the ectopic locations.