Ricardo H. Asch

Surgical Induction of Endometriosis in the Rabbit: Effects on Fertility and Concentrations of Peritoneal Fluid Prostaglandins *

The association of endometriosis and infertility is well know. However, the exact mechanisms involved remain unclear. To study the effect of endometriosis on fertility, an experimental model was developed using New Zealand White rabbits. Endometrium obtained from one uterine horn was surgically implanted into the peritoneum, and the viability of the implants was demonstrated histologically. Adipose tissue was implanted in another group of animals which served as a control. The induction of endometriosis significantly impaired fertility rates (25%) as compared with the control group (75%). The decrease in fertility was independent of adhesion formation and primarily due to a defect in ovulation; however, a postovulatory effect cannot be excluded. Peritoneal fluid prostaglandin (PG) F concentrations increased significantly after the induction of endometriosis, whereas PGE concentrations and the PGF:PGE ratio showed no change. There was no change in PGF or PGE levels in the control group. The increased PGF in peritoneal fluid may alter follicular rupture, ovum transport, corpus luteum function, or implantation, thus representing a mechanism by which endometriosis may cause infertility.

Etiology of infertility in monkeys with endometriosis: luteinized unruptured follicles, luteal phase defects, pelvic adhesions, and spontaneous abortions *

To elucidate the etiology of infertility due to endometriosis, we autografted endometrial or adipose tissue to the pelvic peritoneum of 21 cynomolgus monkeys. These primates were divided into five groups: control animals with adipose tissue autografts (n = 5), animals with microscopic endometriosis (n = 5), animals with mild endometriosis (n = 5), animals with moderate endometriosis (n = 4), and animals with severe endometriosis (n = 2). During three subsequent menstrual cycles, each animal underwent (1) serial assay of peripheral serum gonadotropins and steroids; (2) mating timed according to daily serum 17 beta-estradiol; and (3) laparotomy to document an ovulatory stigma. The chemical and term pregnancy rates were lower among monkeys with moderate or severe endometriosis, as compared with control animals. The impaired fertility in monkeys with endometriosis appeared to be mediated primarily by failure of follicular rupture and/or pelvic adhesions.

Danazol binding and translocation of steroid receptors

Danazol, an isoxazol derivative of ethinyl testosterone which suppresses gonadotropin levels and acts as a weak androgen, is shown by competition studies to bind rat androgen receptor (Ki 10(-8M) and progestin receptor (Ki 10(-7)M) but not estrogen receptor. Effective antigonadotropin doses to the rat in vivo translocate only androgen receptor to target cell nuclei; nuclear receptor levels remain elevated more than 6 hours. The same translocation occurs when rat uteri are incubated with danazol in vitro, showing that the action of danazol is direct and probably does not require metabolic conversion of the drug.

Successful in vitro fertilization and embryo transfer in cynomolgus monkeys.

We have started an in vitro fertilization program in cynomolgus monkeys in an effort to develop an appropriate animal model to improve our knowledge of early embryonic development. In 16 of 25 animals treated with menopausal gonadotropins, preovulatory follicles developed. Follicular aspiration was performed at laparotomy after human chorionic gonadotropin injection. A total of 299 follicles were aspirated, and 251 oocytes were recovered. Oocytes were cultured in 1 ml of growth medium or 100 microliter droplets of medium under mineral oil. Semen samples were obtained by electroejaculation, and the oocytes were inseminated 4 to 24 hours after aspiration. Culture under mineral oil significantly increased the fertilization and cleavage rates. Of 68 embryos produced, 24 have been used in 10 embryo transfers, resulting in two pregnancies.

Interactions of Δ9-Tetrahydrocannabinol (THC) with Hypothalamic Neurotransmitters Controlling Luteinizing Hormone and Prolactin Release

The effects of delta 9-tetrahydrocannabinol (THC) on hypothalamic norepinephrine (NE) and dopamine (DA) turnover and hypothalamic serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA) and LHRH content preceding and during a progesterone- (P) induced LH and prolactin (PRL) surge were investigated in ovariectomized estrogen-primed rats. THC had no effect on basal LH levels, but it inhibited basal PRL levels and blocked the surges of both LH and PRL. The turnover of NE, as estimated by measuring NE depletion after inhibition of tyrosine hydroxylase with alpha-methyl tyrosine (250 mg/kg), in both the anterior (AH) and medial basal hypothalamus (MBH) was significantly inhibited by THC. THC did not significantly affect AH or MBH DA or 5-HT content nor MBH-DA-turnover. Hypothalamic LHRH levels were significantly elevated 4 h after THC administration as compared to the vehicle-injected controls, but pituitary response to exogenous LHRH was not affected. These data suggest that THC inhibits the steroid-induced positive feedback release of LH by reducing NE metabolism and the release of hypothalamic LHRH. Although the mechanism for the inhibition of PRL release by THC is not clear from these experiments, it does not appear that alterations in DA turnover are a contributing factor.

Pelvic Adhesions Following Microsurgical and Macrosurgical Wedge Resection of the Ovaries

The incidence of postoperative adhesion formation following microsurgical and macrosurgical ovarian wedge resection was contrasted in 10 adult female rhesus monkeys. Bilateral wedge resection was performed on day 10 of the luteal phase using microsurgical technique on one ovary and macrosurgical technique contralaterally. Animals were examined 4 weeks postoperatively. Adhesion formation occurred in only one ovary in which microsurgery had been employed (10%). In contrast, adhesion formation followed macroscopic ovarian wedge resection in five ovaries (50%). All adhesions were periovarian, emanating from the suture line on the ovarian surface. Adhesions were most common on the nonovulatory ovary.

Sperm washing and swim-up technique using antibiotics removes microbes from human semen

Pelvic infections may follow intrauterine or intratubal insemination with washed semen. In this study, we determined whether sperm washing removes microorganisms from human semen. Before and after semen wash, we cultured 15 ejaculates for aerobic and anaerobic bacteria, genital mycoplasma, and chlamydia. All semen samples had from one to five organisms isolated (total, 40 isolates) before the semen wash preparation. The mean number (+/- standard deviation) of isolates per sample was 2.67 +/- 1.35. After the semen were prepared, none of the samples showed a positive culture. The decrease in the number of samples with positive cultures and the decrease in the number of isolates is significant (P less than 0.0001). After sperm washing, electronmicroscopic studies did not show any microbes attached to any portion of the spermatozoa. We conclude that the method of sperm wash preparation used is effective in removing microbes present in human semen.

Interactions of cocaine and Δ9-tetrahydrocannabinol with the hypothalamic-hypophysial axis of the female rat

The acute effects of cocaine and/or delta 9-tetrahydrocannabinol (THC) were studied in ovariectomized female rats. Intermediate doses of cocaine (10 or 20 mg/kg) caused an increase in serum luteinizing hormone (LH) and a decrease in serum prolactin levels, whereas a higher dose (40 mg/kg) caused a decrease in serum LH. THC (10 mg/kg) attenuated serum LH and prolactin levels. The THC effect on LH was blocked by cocaine. Neither drug alone or in combination affected serum follicle-stimulating hormone levels. The cocaine-induced changes in LH levels were closely paralleled by changes in hypothalamic norepinephrine content, suggesting a neurochemical basis for cocaines action on LH release.

Precoital Single Doses of Δ9-Tetrahydrocannabinol Block Ovulation in the Rabbit

delta9-Tetrahydrocannabinol (delta9-THC) inhibits pituitary gonadotropin secretion in castrated rhesus monkeys. Also, delta9-THC blocks the ovulatory reflux in rabbits. We report now the dose-response relationship of precoital single doses of delta9-THC on luteinizing hormone (LH) release and ovulation in the rabbit. Forty-five female rabbits in estrous were divided into nine groups of five animals. Groups 1 to 5 received a single intramuscular dose of delta9-THC (5,2.5, 1.25, 0.612, and 0.306 mg/kg, respectively) 2 hours before mating; animals of group 6 received vehicle only. In animals of groups 7 and 8 ovulation was induced with 100 IU of human chorionic gonadotropic (hCG), given intravenously 2 hours after the administration of delta9-THC (5 mg/kg) or vehicle. Rabbit luteinizing hormone (rLH) was measured in plasma 90 to 120 minutes after coitus or hCG administration. After the injection of 5 mg of delta9-THC, luteinizing hormone-releasing factor (LH-RF) (20 microgram intravenously) was administered to the animals of group 9. All animals of groups 6, 7, and 8 ovulated. A dose-response curve was observed in the animals treated with delta9-THC and natural mating. Whereas none of the animals treated with 5 or 2.5 mg/kg ovulated, one of the group treated with 1.25 mg/kg, two of the group treated with 0.612 mg/kg, and all treated with 0.312 mg/kg ovulated. Ovulations correlated with postcoital levels of rLH. All animals of group 9 ovulated, indicating that the site of action of delta9-THC is suprapituitary, probably hypothalamic.

Mechanism of induction of luteal phase defects by danazol.

The effects of danazol on the luteal phase of cycling rhesus monkeys and on the ability of the intact rhesus monkey to respond to hCG stimulation were studied in two experiments. In a third experiment the impact of danazol upon the response of the decapsulated mouse testis to hCG stimulation was evaluated. These experiments demonstrated that danazol shortens the luteal phase and decreases progesterone production in intact monkeys via a direct effect on the gonad.