Jose P. Balmaceda

Relationship of weight to successful induction of ovulation with clomiphene citrate

Over a 6 1/2-year period, 117 patients who were anovulatory, euthyroid, and estrogen-primed were treated with clomiphene citrate. Graduated doses from 50 mg to 250 mg daily for 5 days were used to induce ovulation. Of 62 patients who completed treatment, 50 ovulated and 12 did not. Several factors, including age, duration of infertility, weight, previous menstrual history, previous pregnancy history, and previous use of oral contraceptives, were investigated to determine conditions which might influence response. Only weight was found to be significantly different between responders and nonresponders. Furthermore, there was a linear relationship between body weight and dose of clomiphene required to induce ovulation. The ovulation rate for those completing therapy was 81% with a pregnancy rate of 76% of the total and 94% of those ovulating. Population homogeneity with anovulation as the major cause of infertility appears to be the most plausible explanation for the high pregnancy rate.

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.

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.

Embryo cryopreservation in cynomolgus monkeys

An improved knowledge of cryopreservation of primate embryos will have important research and clinical application. Fifty-six 4- to 8-cell in vitro fertilized embryos were frozen in HEPES-buffered Tyrodes solution containing 1.5 M dimethylsulfoxide (DMSO) and cooled at the rate of 0.3 degrees C/minute to -39 degrees C before being transferred into liquid nitrogen. Embryos were rapidly thawed at room temperature for 2 minutes. DMSO was diluted with medium in three steps at 5-minute intervals. Of the 56 embryos, 39 (70%) were classified as viable on the basis of surviving the freezing process with greater than 50% of their blastomeres intact. Twelve of the 39 embryos were cultured overnight, and 11 cleaved at least once. Twenty-five embryos were transferred to nine synchronized, unstimulated recipient monkeys 24 to 48 hours after ovulation. Three pregnancies (33.3%) resulted from the nine transfers.

The effects of chronic administration of LH-RH agonists and antagonists on the menstrual cycle and endometrium of the rhesus monkey

Regularly cycling rhesus monkeys (Macaca mulatta) were used to study the effects of prolonged administration of LH-RH analogs on the menstrual cycle and the endometrium. According to the treatment, animals were divided into: Group 1, vehicle; Group 2, LH-RH agonist (D-Trp6 LH-RH, 20 micrograms/day); and Group 3, LH-RH antagonist [( N-Ac-D-Trp1,3, D-p-Cl-Phe2,D-Arg6,D- Ala10 ]-LH-RH,200 micrograms/day) for 90 days. Follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and progesterone (P) were measured every second day until thirty days past the discontinuation of drug administration. Endometrial biopsies were obtained on days 10, 40, 90 and 120 and processed for histologic exam and determination of estrogen (E) and progesterone receptors. Animals of Group 1 presented regular cycles, while those in Groups 2 and 3 remained anovulatory throughout the treatment. Animals of Group 2 presented different degrees of endometrial hyperplasia during treatment and animals of Group 3 showed either resting or atrophic endometrium. Administration of LH-RH agonist produced a marked increase in E and P endometrial receptors and the antagonist produced a decrease in P receptors. In both instances, reversal of the effects on the menstrual cycle and in the endometrium was observed 30 days after discontinuation of drug administration.

Failure of seminal plasma to enter the uterus and oviducts of the rabbit following artificial insemination.

Twelve adult does in estrus were artificially inseminated with 1 ml of semen from bucks that had received injections of 3H-thymidine 22 to 32 days previously. The ejaculates contained radioactive seminal plasma and unlabeled sperm. The reproductive tracts of the does were removed at selected intervals after insemination, divided into segments of no more than 300 mg, and processed for scintillation counting. Significant levels of radioactivity were never detected in any uterine or oviductal segment. The technique was capable of detecting as little as 1.5 X 10(-4) ml of semen in a segment. These results demonstrate that seminal plasma is not transported from the vagina into the uterus or oviducts in the rabbit, emphasizing the importance of the motility of spermatozoa in their passage to the site of fertilization.

Prostaglandin Production by Rhesus Monkey Corpora Lutea in Vitro: Effects of Estrogen Administration

Luteolysis was induced in normal cycling rhesus monkeys (Macaca mulatta) by daily administration of estradiol cypionate (50 μg/kg) from day 1 to 7 postovulatory (controls received vehicle). Luteectomy was performed after treatment, using microsurgical techniques and minimal manipulation. Serum progesterone concentrations did not demonstrate characteristic luteal rise in estrogen‐treated animals as compared to controls. Estrogen treatment increased prostaglandin F2α/ E ratio produced in vitro by the corpora lutea twofold compared to controls. These results support our previous observations that luteolysis in the rhesus monkey is associated with increased production of prostaglandin F2α and decreased production of prostaglandin E by the corpus luteum.

Gamete intrafallopian transfer vs superovulation with intrauterine insemination for the treatment of infertility

Superovulation with intrauterine insemination (SO-IUI) has been suggested as an alternative to gamete intrafallopian transfer (GIFT), despite the absence of controlled or comparative trials. We retrospectively analyzed all GIFT and SO-IUI cycles performed concurrently from January 1985 to August of 1987 at a single university center. Pregnancy rates were significantly better for GIFT than SO-IUI (P<0.001), with an odds ratio of 3.25 (P=0.001). Stepwise multiple logistic regression identifield factors that correlate with pregnancy: absence of endometriosis (P=0.05), infertility<3 years duration (P=0.002), TMS ≧30×106 (P=0.005), and treatment with GIFT rather than SO-IUI (P=0.001). These data give a first approximation of the increased efficacy of GIFT versus SO-IUI and provide valuable insight into significant confounding variables to be considered when planning a randomized, prospective trial to evaluate these techniques.