Claudio Chillik

The role of in vitro fertilization in infertile patients with endometriosis

Thirty-nine cycles were studied in patients with a history of endometriosis who went through in vitro fertilization. In 15 cycles, there was no evidence of endometriosis; in 10 cycles, the patients had mild or moderate disease; in 14 cycles, severe or extensive endometriosis was found. The pregnancy rates per cycle were 33%, 60%, and 7%, respectively (groups I and II, no significant difference; groups II and III, P less than 0.01). The difference was due to the different number of oocytes aspirated at laparoscopy because of technical problems in the cases with severe and extensive disease. There was also a significant difference in the number of pregnancies per transferred cycles. There was no difference in the luteal phase in the three groups. The reproductive potential, which seemed to be similar in groups I and II, was severely impaired in the group with severe endometriosis.

Contraceptive potential of RU 486 by ovulation inhibition: I. Pituitary versus ovarian action with blockade of estrogen induced endometrial proliferation

In previous studies, RU 486 administration arrested spontaneous folliculogenesis. To investigate the central versus peripheral effects of RU 486 on the ovarian/menstrual cycle, including endometrial proliferation, RU 486 was administered daily (10 mg/kg/day, im) from menstrual cycle day 3 or 7 to day 25 in normal adult cynomolgus monkeys receiving hMG treatment (37.5 IU/day) from days 3-8 (n = 6). RU 486 administration with hMG/hCG therapy did not inhibit ovarian response, as evidenced by steroidogenesis and ovulation. Nine of 23 oocytes retrieved by lavage or follicular aspiration at laparotomy after ovulation induction were morphologically classified as mature preovulatory status. Whereas an endometrial biopsy performed on cycle day 25 in control monkeys revealed an in phase mature secretory endometrium, histologic sections from RU 486 plus hMG/hCG treated females uniformly demonstrated atrophic to weakly proliferative endometrium on cycle day 25, despite serum estradiol levels greater than 300 pg/ml. Three months after the initial 25-day study endometrial biopsies revealed persistent atrophic endometrium, even though repeated ovulation induction with hMG/hCG therapy elevated serum estrogen concentrations. The findings prevailed whether RU 486 treatment began on cycle day 3 or 7. The intermenstrual interval was significantly (P less than 0.01) lengthened by RU 486 treatments (28.5 +/- 2.0, control vs 131.3 +/- 11.5 days, RU 486). In summary, RU 486 consistently blocked ovulation unless hCG was provided and elicited a persistent retardation of early proliferative endometrium when administered daily beginning in early or mid-follicular phase. The normal mitogenic effects of elevated ovarian estrogen secretion on endometrial tissue were quelled, uniformly resulting in amenorrhea. The long-lasting action of RU 486, causing ovulation inhibition and atrophic endometrium, may be due to the depot effect of im injection. In addition, RU 486 did not prevent ovarian steroidogenesis, ovulation or oocyte maturation when an ovulation induction regimen of hMG/hCG was given. These findings show that RU 486 prevented ovulation by diminishing pituitary gonadotropin secretion, rather than by direct effects on ovarian folliculogenesis, and induced amenorrhea by inhibiting estrogen-induced endometrial proliferation.

Tolerance of perinidatory primate embryos to RU 486 exposure in vitro and in vivo

Monkey embryos were exposed to RU 486 both in vitro and in vivo (with and without progesterone therapy) in the perinidatory interval. These primate embryos were highly tolerant of RU 486, except when RU 486 alone terminated early pregnancy. There were no indications of teratogenicity in this limited trial when the embryos were exposed to RU 486 either before implantation (10(-7)M in 24-hour cultures) or during the immediate post-implantation interval (50 mg orally/day; days 32 to 39 LMP).

Characterizing pituitary response to a gonadotropin-releasing hormone (GnRH) antagonist in monkeys: tonic follicle-stimulating hormone/luteinizing hormone secretion versus acute GnRH challenge tests before, during, and after treatment *

Pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) challenge test before, during, and after GnRH antagonist administration was compared in four ovariectomized female monkeys receiving GnRH antagonist intramuscularly (IM) at increasing doses of 0.3, 1.0, and 3.0 mg/kg/day over 9 days. Three days before and 3 days after treatment, monkeys received vehicle alone. On experiment days 4, 7, 10, 13, and 16, 100 micrograms of GnRH was administered intravenously (IV) and blood drawn at 0 and 30 minutes. Before treatment, tonic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were 248 +/- 105 and 178 +/- 31 ng/ml, respectively; after 0.3 mg/kg/day of GnRH antagonist, FSH and LH decreased to 30 +/- 6 and 41 +/- 4 ng/ml, respectively. After treatment with either 1 mg/kg/day or 3 mg/kg/day of GnRH antagonist, both gonadotropins were undetectable in serum. Monkeys with lower initial levels of gonadotropins were suppressed by 48 hours after GnRH antagonist, while those with higher tonic gonadotropins were suppressed 6 days later (FSH: r = 0.992; LH: r = 0.833). The data show that initial physiologic status is predictive of the rapidity of the suppression response induced by a GnRH antagonist and that, after achieving pituitary suppression, responsivity to an IV GnRH challenge test may be restored before normal tonic FSH/LH secretion is regained.

The role of LHRH agonists and antagonists.

Administration of GnRH analogues (agonists as well as antagonists) produces suppression of the pituitary---gonadal axis, thus inhibiting the secretion of LH, FSH and sexual steroids. For this reason, analogs are indicated in all those clinical situations where suppression of gonadotrophins (precocious puberty, contraception) or of sexual steroids (endometriosis, prostate hyperplasia, cancer, uterine fibroids) is desired. For several years GnRH agonists have been used in combination with gonadotrophins for ovarian stimulation for assisted reproduction in order to control premature LH surges and to reduce cancellation rate with improvement of the pregnancy rate per cycle. This effect is obtained after 2 weeks of agonist administration. The immediate suppression of the pituitary achieved by GnRH antagonists without an initial stimulatory effect is the main advantage of these compounds over the agonists. The prevention of a premature LH surge by GnRH antagonists can be obtained by multiple dose or by a single administration. Both protocols offer the following advantages over the agonists: they require fewer ampoules of gonadotrophins, shorter duration of stimulation, there is a preserved pituitary response to GnRH, less risk of ovarian hyperstimulation syndrome and the luteal phase seems to be more preserved. The main disadvantages of the antagonists are that they are expensive and that pregnancy rate appears to be slightly lower than with the agonists. GnRH antagonists will probably replace agonists in ovarian stimulation treatment for assisted reproduction techniques.

RU486-induced menses in cynomolgus monkeys: uniformity of endometrial sloughing

A progesterone (P) antagonist (RU486) was administered to cynomolgus monkeys in the midluteal phase (cycle day 21 to 24), alone or in combination with P. Vehicle only was administered to other monkeys that served as controls. On cycle day 25, hysterectomy was performed on all of the monkeys. The endometrium was studied histologically in fundal, medial, and isthmic regions. RU486 induced menses in all treated monkeys; but the amount of residual endometrium was slightly higher in primates that received RU486 plus P. The effect of RU 486 on the endometrium was surprisingly homogeneous throughout the uterus. The findings demonstrate that administration of RU486 in the luteal phase induced a nearly uniform and homogeneous sloughing of the endometrium. Whereas concurrent P treatment did not prevent induction of menstruation, the depth of endometrial shedding was less than with RU486 alone.

IN VITRO FERTILIZATION AND THE MALE FACTOR

In vitro fertilization (IVF) followed by embryo transfer (replacement) to the patient donating the female gametes and using the husband’s sperm for fertilization purposes is the classic form of therapy, devised in the 197Os, to overcome the problem of female infertility due to irreparable tubal damage.’ As experience increased with the procedure, several other indications were found for this therapeutic modality, namely, in the female the presence of endometriosis which does not respond to the commonly used treatment protocols, severe cervical factors that cannot be overcome by intrauterine insemination, the presence of systemic or local antisperm antibodies as the only detectable factor for infertility, and the female who has no identifiable factors for her reproductive problem and who has a normal husband, but who nevertheless cannot achieve pregnancy (infertile normal couple). Soon it was realized that IVF needed a small amount of sperm (as low as perhaps 12,000) for fertilization to occur. Therefore, it was immediately inferred that this procedure may also be indicated in infertility due to male factor. Several groups started working with this type of patient, trying to correct the fertility problem in males in whom the commonly accepted treatment protocols had already failed. Other forms of assisted reproduction have been developed as variants of the classic type of IVF with embryo replacement (gamete intrafallopian transfer, gamete donation from male

Transplacental passage of a progesterone antagonist in monkeys

The progesterone antagonist RU 486 dramatically increases myometrial contractility of the pregnant uterus, making it a potential adjunctive therapy for labor induction or therapeutic pregnancy termination. Sixteen female cynomolgus monkeys were studied during the second or third trimester of pregnancy. Hysterotomies were performed with the animals under anesthesia, providing access to the intact placental vasculature. RU 486 (25 mg) was injected intravenously into the mothers. Serial blood samples were drawn from the maternal and fetal-placental compartments for a period of 2 hours. RU 486 achieved a gradient equilibrium between the maternal and fetal-placental circulation within 5 minutes, suggesting free passage by simple diffusion. The clearance kinetics of immunoreactive RU 486 are consistent with an open three-compartment system in mother and fetus. The fetal-placental index decreased from 31.2% to 17.8% between the second and the third trimester of pregnancy. There was no acute toxicity of the RU 486 noticed during the experimental course.

Pronuclear abnormalities and cytoskeletal organization during assisted fertilization in a patient with multifollicular ovarian response.

AbstractPurpose: To analyze the distribution of α tubulins and acetylated α tubulins and the chromatin configuration in abnormally fertilized zygotes from a patient with a multifollicular ovarian response after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Methods: Immunofluorescence and phase contrast microscopy was performed in abnormally fertilized zygotes. Results: After phase contrast microscopy analysis, immunofluorescence staining was performed in 20 oocytes that developed ≥3 pronuclei (PN) and karyomeres after IVF–ICSI. Around 80% of the abnormal zygotes from IVF were the consequence of monospermic fertilizations. Retention of the second polar body (PB) and the presumptive split of ≥1 PN within the cytoplasm were the main events present in most oocytes after IVF–ICSI. Conclusions: Fluorescence labeling of selected sperm and oocyte components affords a unique view of abnormal fertilized zygotes. Surprisingly, anomalies detected after IVF–ICSI showed similar etiologies in this special group of zygotes.

Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm**Presented in part at the 35th Annual Meeting of the Society for Gynecologic Investigation, Baltimore, Maryland, March 17 to 20, 1988.††Supported in part by a Barlex Scholar Award, New York, New York.

Approximately one fourth of all human oocytes collected for in vitro fertilization are of immature origin. Even when these oocytes undergo nuclear maturation, fertilization, and cleavage in vitro, transfer of such embryos rarely results in pregnancy reaching delivery. We hypothesized that human embryos derived from prophase I oocytes were developmentally incompetent because they lacked a factor(s) found in in vivo matured oocytes. Using micromanipulation techniques in monkeys, we removed ooplasm from metaphase II oocytes and injected it into prophase I oocytes. After nuclear maturation, oocytes were transferred to the fallopian tube for fertilization. After ooplasmic transfusion, prophase I oocytes resulted in a delivery rate of 13%. When metaphase II ooplasm was heated or exposed to ribonuclease A before microinjection into prophase I oocytes, it lost effectiveness in conferring developmental competence.