Successful human pregnancy following in vitro fertilization using frozen semen.

Frozen-thawed husband's semen was utilized for insemination of spouse's oocytes in vitro. Postthaw semen had a low motility (40%) with a poor forward progression (+2), which subsequently decreased rapidly in the regularly used Ham's F-10 medium (pH 7.42, 280 mosmol/kg) supplemented with 7.5% fetal cord serum. When Ham's F-10 was supplemented with an additional 0.5 mmol each of magnesium sulfate (MgSO4) and potassium bicarbonate (KHCO3) at a higher pH (pH 7.69, 280 mosmol/kg), the sperm maintained their motility and forward progression and fertilized all the oocytes, which subsequently cleaved and developed into normal embryos. Transfer of these embryos resulted in a normal pregnancy. Our results indicate that cryopreserved semen of poor quality can be successfully employed for an in vitro fertilization-embryo transfer (IVF-ET) program, especially when MgSO4 and KHCO3 are used in Ham's F-10 at a higher pH.

A comprehensive dose-response study of clomiphene citrate for enhancement of the primate ovarian/menstrual cycle.

Despite the increasing use of clomiphene citrate (CC) in normally cycling women undergoing in vitro fertilization, comprehensive data on the dose-response effects of the drug are unavailable. Twenty-four adult, normally cycling monkeys were given CC on cycle days 5 through 9 in doses ranging from 1 mg/kg to 10 mg/kg. Serum estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone were measured daily. CC stimulated an early, attenuated rise in serum LH which correlated with subsequent follicle development. Concurrently, serum E2 rose to preovulatory levels. No significant increase in serum FSH concentration was seen as a result of CC therapy. No correlation was seen between CC dose and peak levels of E2, LH, or FSH. The ovulatory status of the treatment cycles was also independent of dose. The factors that modulate these hormonal changes may involve direct effects of CC, estrogen feedback, and/or ovarian factors as yet uncharacterized. The individual variation of ovarian response, however, appears to be independent of the dose of CC.

Medical hypophysectomy: I. Dose-response using a gonadotropin-releasing hormone antagonist.

The hypothalamic-pituitary-ovarian axis can be "dissected" in a nonsurgical and reversible fashion by the administration of a potent gonadotropin-releasing hormone (GnRH) antagonist. We created a transient, functional lesion at the level of the pituitary gonadotrope by using a potent GnRH antagonist ([ Ac- pClPhe1 , pClDPhe2 , DTrp3 , DArg6 , DAla10 ]-GnRH). In long-term castrate cynomolgus monkeys, doses of 0.05 to 2.0 mg/kg/day intramuscularly were administered for a total of 32 days. At doses up to 0.2 mg/kg/day, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in circulation were only moderately suppressed; these subjects responded to an estradiol challenge by manifesting an LH elevation or surge within 48 hours. At doses of 0.5 to 1.0 mg/kg/day, FSH and LH secretion was suppressed to or below the limits of assay detection within 7 days, remaining in a severely hypogonadotropic state for the remainder of the treatment interval. Using 2 mg/kg/day, estradiol-positive feedback for midcycle-like LH/FSH surges was fully inhibited. This suppression of gonadotropin secretion was rapidly reversible, in that circulating gonadotropin levels had returned to pretreatment castrate levels within 60 days after termination of GnRH antagonist treatments. These findings suggest that potent GnRH antagonists can effectively create a hypogonadotropic milieu without the initial enhancement of gonadotropin secretion that occurs during initiation of GnRH agonist therapy. "Medical hypophysectomy" through GnRH antagonist administration may permit a more direct and controlled approach to gonadal therapies such as ovulation induction.

Medical hypophysectomy: II. Variability of ovarian response to gonadotropin therapy.

In an attempt to control individual variability of ovarian response to gonadotropin therapy, ovulatory monkeys received either "pure" follicle-stimulating hormone (FSH) or human menopausal gonadotropin (hMG), with or without gonadotropin-releasing hormone (GnRH) antagonist administration. Among females that responded to gonadotropin therapy, the GnRH antagonist reduced (P less than 0.05) the variability of serum estradiol patterns. Surprisingly, after pretreatment and concurrent administration of the GnRH antagonist, FSH alone was as effective as the FSH/luteinizing hormone (LH) mixture (hMG) in stimulating follicular maturation, even when serum LH levels were at or below the limits of detection. The results indicate that in a rapidly reversible hypogonadotropic state approaching a "medical hypophysectomy," concurrent gonadotropin therapy produces a less varied ovarian response. The relative (un)importance of LH in the primate ovarian cycle seems diminished in the face of evidence that FSH alone, or in the presence of vanishingly small amounts of LH, supports follicular maturation and dynamic estrogen biosynthesis.

Human menopausal gonadotropin stimulation in monkeys: blockade of the luteinizing hormone surge by a highly transient ovarian factor.

Women and monkeys treated with human menopausal gonadotropin (hMG) often fail to demonstrate timely luteinizing hormone (LH) or follicle-stimulating hormone (FSH) surges despite serum estradiol (E2) levels sufficient to elicit positive feedback of LH and FSH secretion. Here we explored the mechanism of this blockade of estrogen-mediated positive feedback by hMG and the duration of this effect in acutely ovariectomized monkeys. Cycling cynomolgus monkeys (n = 14) were administered hMG (22.5 IU, intramuscularly) daily beginning on cycle day 3. Three monkeys received an E2-benzoate challenge on day 10, resulting in peak E2 levels of 892 +/- 313 pg/ml, without subsequent LH or FSH surges. Four monkeys underwent bilateral ovariectomy on day 12, followed within 60 to 90 minutes by gonadotropin-releasing hormone (GnRH) administration (15 micrograms, intravenously). Five intact monkeys underwent a similar GnRH challenge. LH response to GnRH in the intact monkeys was significantly suppressed in comparison with the ovariectomized group. None of the animals manifested an FSH response to GnRH administration. Two additional monkeys did have a spontaneous LH surge during hMG administration. We conclude that hMG stimulates the production of an ovarian factor(s) which blocks the pituitary LH response to GnRH. This blockade of GnRH action on the pituitary may be the mechanism by which hMG stimulation prevents estrogen-mediated positive feedback of LH secretion. The putative ovarian factor(s) has a relatively short circulatory half-life and/or binding time, resulting in loss of its blocking activity within 90 minutes after bilateral ovariectomy. Evidence is presented to suggest that this factor(s), designated gonadotropin surge-inhibiting factor, belongs to an amorphous group of nonsteroidal ovarian hormones that remain to be further characterized.