Robert M. Riehl

Metformin treatment of patients with polycystic ovary syndrome undergoing in vitro fertilization improves outcomes and is associated with modulation of the insulin-like growth factors

OBJECTIVE To determine if metformin therapy improves in vitro fertilization (IVF) outcomes in patients with clomiphene-resistant polycystic ovarian syndrome (PCOS). DESIGN Retrospective data analysis of selective groups of patients. SETTING A private IVF unit. PATIENT(S) Forty-six women with clomiphene citrate-resistant PCOS underwent 60 cycles of IVF embryo transfer with intracytoplasmic sperm injection. INTERVENTION(S) In half of the cycles, patients received metformin (1000 to 1500 mg) daily, starting the cycle prior to gonadotropin treatment. MAIN OUTCOME MEASURE(S) Total number of follicles; serum estradiol (E2) on the day of hCG administration and the cycles E2 maximum; total number of oocytes, mature oocytes, embryos, fertilization, and pregnancy rates; and follicular fluid levels of insulin-like growth factors (IGF-I, IGF-II) and IGF-binding proteins (IGFBP-1, IGFBP-3). RESULT(S) In patients treated with metformin, the total number of follicles on the day of hCG treatment was decreased (23 +/- 1.2 vs. 33 +/- 2.6) with no change in follicles > or = 14 mm in diameter (21 +/- 1.2 vs. 25 +/- 1.7). Metformin treatment did not affect the mean number of oocytes retrieved (22 +/- 1.9 vs. 20.3 +/- 1.5). However, the mean number of mature oocytes (18.4 +/- 1.5 vs. 13 +/- 1.5) and embryos cleaved (12.5 +/- 1.5 vs. 5.9 +/- 0.9) were increased after metformin treatment. Fertilization rates (64% vs. 43%) and clinical pregnancy rates (70% vs.30%) were also increased. Metformin led to modulation of preovulatory of follicular fluid IGF levels with increases of IGF-I (140 +/- 8 vs. 109 +/- 7ng/mL) and decreased of IGFBP-1 (133 +/- 8 vs.153 +/- 9ng/mL). CONCLUSION(S) Metformin use appears to improve IVF outcomes in patients with clomiphene citrate-resistant PCOS.

Cocaine Disrupts Estrous Cyclicity and Alters the Reproductive Neuroendocrine Axis in the Rat

Although a common drug of abuse, cocaines effects on cyclic reproductive functions and the neuroendocrine systems regulating these functions have not been studied. Here, we report the effects of cocaine on (1) estrous cyclicity and ovulation rates and (2) the stimulated in vitro release of hypothalamic GnRH and aminergic neurotransmitters directly involved in regulating or modulating GnRH release. Within 7 days of treatment with 10 mg kg-1 day-1 of cocaine HCl subcutaneously, rats demonstrated significant estrous cycle irregularity including repetitive days of estrus and prolonged periods of diestrus. After 6 weeks of treatment, cocaine-treated rats exhibited a 44.3% decrease in ovulation rates. For the in vitro studies, bilaterally ovariectomized rats were injected with cocaine (10 mg kg-1 day-1) or with saline for 2 weeks. Each rat received estradiol benzoate (50 mg kg-1 day-1 s.c.) for 2 days before sacrifice. Hypothalamic slices were prepared, placed in 0.1 ml microchambers and perfused with modified Krebs buffer (pH 7.4) using a programmable perfusion system. Basal release of norepinephrine (NE) and serotonin (5HT) was significantly increased in the cocaine-treated group versus controls. Ten-minute pulses of 10(-7)M progesterone (P4) increase NE and 5HT, but not dopamine (DA), release in the saline-treated group. In contrast, pulses of P4 increased NE, but not 5HT or DA, in the cocaine-treated rats. Ten-minute pulses of 0.1 microM NE increased GnRH release in both saline- and cocaine-treated rats. However, the response to pulsed NE was significantly attenuated in the cocaine-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

c-myc protooncogene polypeptide expression in endometriosis.

The c-myc protooncogene and its polypeptide product are important regulators of cell proliferation and differentiation, and ovarian steroids are believed to stimulate growth of various uterine cell types through altered expression of the c-myc gene. To determine whether c-myc expression may also be involved in the growth and development of endometriosis, we assessed c-myc expression in eutopic and ectopic endometrial tissue obtained from women undergoing surgery for endometriosis. Immunocytochemistry using a monoclonal antibody to the c-myc protein demonstrated positive staining of glandular and stromal cell nuclei, and cytoplasmic staining of glandular but not stromal cells in both eutopic and ectopic endometrium. These findings suggest that c-myc expression may be an important regulator of cell proliferation in endometriotic tissue.

A simple method for preparing single cell suspensions of heart and smooth muscle for radioreceptor labeling studies

A simple method of preparing single cell suspensions for radioligand binding studies is described. The method involves incubating tissues in the presence of collagenase and elastase for 90 min in physiological solution with 1 mM calcium chloride and the mechanical disruption of the tissue by pipetting. The tissues examined were atria, ventricle, bladder, uterus, and taenia coli removed from mature guinea pigs. Viability of the cells by trypan blue exclusion showed 60-88% viable cells and receptor binding studies using (3H]-1-quinuclidinyl benzilate (QNB) yield KD values of approximately equal to 0.1 nM. The receptor numbers for each tissue were (receptors/cell): atria, 5700; ventricle, 11,000; uterus, 31,000; bladder, 44,000; taenia coli, 68,000.

Gonadotropin-releasing hormone antagonists attenuate estrogen/progesterone-induced hyperprolactinemia in monkeys

Previous studies have documented that exogenous gonadotropin-releasing hormone (GnRH) stimulates prolactin (PRL) secretion and augments thyrotropin-releasing hormone-induced PRL release. Further, the concomitant pulsatile release of PRL and luteinizing hormone (LH) suggests that GnRH may be an important regulator of PRL release in certain physiologic states. The authors explored this possibility by evaluating the effect of a GnRH antagonist ([Ac-pClPhe1, pClPhe2, DTrp3, DAla10]-GnRH; GnRH-antagonist) on PRL secretion in monkeys with induced hyperprolactinemia. Monkeys were given estradiol (E2) benzoate 25 mg/kg intramuscularly (IM) on cycle days 1 to 28, and a 3-cm progesterone (P) silastic capsule was placed on cycle day 15 and removed on day 28. On cycle days 15 to 28, monkeys were given IM injections of 1 mg/kg GnRH-antagonist (n = 3), 2 mg/kg GnRH-antagonist (n = 3), or vehicle (n = 3). Daily blood samples were assayed for E2, P, and PRL. The degree of PRL elevation was calculated as percent increase in area under the curve for days 15 to 28 when compared with days 1 through 14 (baseline). Luteinizing hormone levels were calculated similarly. Results indicate a dose-dependent effect of GnRH-antagonist on PRL secretion, with the larger dose producing a significantly lower hyperprolactinemic response, as well as a decline in LH. Thus, GnRH-antagonist attenuates induced hyperprolactinemia in a dose similar to that which suppresses LH release. These findings suggest that GnRH is a physiologic regulator of pituitary PRL secretion. In addition, GnRH analogs may be of benefit in controlled ovarian hyperstimulation by attenuating gonadotropin-induced hyperprolactinemia, thereby reducing potential adverse effects on fertility.

Effects of clomiphene citrate and leuprolide acetate on luteal-phase hyperprolactinemia during ovarian stimulation with menopausal gonadotropins

Hyperprolactinemia, a known modulator of reproductive function, occurs commonly in women undergoing ovarian stimulation with human menopausal gonadotropins (hMG). Clomiphene citrate (CC) and gonadotropin releasing hormone analogues (GnRHa), when administered during the luteal phase, attenuate the hyperprolactinemic response to hMG. We asked whether follicular-phase administration of CC and GnRHa, as employed clinically in women undergoing ovarian stimulation forin vitro fertilization or gamete intrafallopian transfer, would alter the incidence and severity of hMG-induced luteal-phase hyperprolactinemia. Seventy-five percent of all patients had at least one luteal prolactin level >25 ng/ml, and 40% had mean luteal-phase prolactin levels >25 ng/ml. The incidence of hyperprolactinemia was similar in pregnant and nonpregnant cycles. The incidence of hyperprolactinemia was similar for both the GnRH agonist-treated group and those given clomiphene citrate. The increase in mean luteal prolactin levels over the follicular-phase baseline level was significantly greater in the CC-treated group (P=0.03). This was due to the significant suppression of follicular-phase baseline prolactin levels in patients receiving CC. We conclude that neither CC nor GnRHa administration in the follicular phase prevents lutealphase hyperprolactinemia in women undergoing ovarian stimulation with hMG.

The effects of environmental stress on steroid receptor levels and steroid-induced morphogenesis in Achlya ambisexualis.

Incubation of Achlya ambisexualis at elevated temperature (heat shock) or in the presence of sodium arsenite resulted in an inhibition of steroid hormone-induced responsiveness. The effect of heat shock was time- and temperature-dependent and more severe than the effect of sodium arsenite. Incubation at 37 degrees C for 30 min completely abolished the steroid-induced response and full recovery was not observed until 6 h after a return to the normal growth temperature of 22 degrees C. Heat shock and arsenite treatment had no effect on the cellular uptake of the steroid hormone, but heat shock resulted in a time- and temperature-dependent loss in the cellular level of steroid receptors. In contrast, arsenite treatment had little effect on the concentration of steroid receptors. However, both heat shock and arsenite treatment produced a long-term (4 h) and transient (1 h) inhibition of total protein synthesis, respectively. The recovery of steroid-induced responsiveness following heat shock was observed after both protein synthesis and steroid hormone receptor levels had returned to normal values.

Effects of tamoxifen on corpus luteum function and luteal phase length in cynomolgus monkeys

Previous data in nonhuman primates have demonstrated that tamoxifen prolongs the luteal phase without altering reproductive hormone levels. A small study in humans found no effect on menstrual cycle length, but an increase in luteal ovarian steroid levels. In view of these conflicting results, we studied the effect of tamoxifen on corpus luteum (CL) function in monkeys (n = 20). Blood was obtained daily beginning cycle day 8, and sera assayed for estradiol (E2), progesterone (P), luteinizing hormone, and follicle-stimulating hormone. Four days after the midcycle E2 peak, laparoscopy confirmed CL formation, and the animals were administered (1) lactose (n = 7), (2) tamoxifen, 0.5 mg.kg-1.d-1 (n = 6), or (3) tamoxifen, 3.0 mg.kg-1.d-1 (n = 7) for 12 consecutive days. Serum collection continued until cycle day 50 or menses, whichever came first. Results indicate a biphasic response among tamoxifen-treated animals, with 7 of 13 developing prolonged luteal phases. There was, however, no significant difference in luteal phase length among the three groups, although when the two groups given tamoxifen were combined, the difference in luteal phase length versus controls approached significance. No differences were found among peak P levels, mean luteal phase P levels, or mean luteal phase gonadotropin levels. No variables were found to correlate significantly with luteal phase length. These results suggest that luteal phase administration of the antiestrogen tamoxifen does not alter pituitary gonadotropin secretion or CL function. However, tamoxifen does prolong luteal phase length in a subset of monkeys, perhaps via a direct effect on the endometrium.