A pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage.

BACKGROUND: In the present report we analyse the structural and functional features of sperm from a patient with severe asthenoteratozoospermia and failure of cleavage after ICSI. METHODS: Sperm were studied by phase contrast and transmission electron microscopy and microinjected into bovine oocytes to examine aster formation using antibodies against acetylated alpha- and beta-tubulins. RESULTS: Acephalic sperm, headless tails and abnormal alignments of the head-tail junction were observed. Flagella evidenced the features of dysplasia of the fibrous sheath. Bovine oocytes injected with patient's sperm showed male and female pronuclei but a faulty development of microtubules from the sperm-derived centrosome. The first ICSI attempt using conventional sperm selection methods resulted in fertilized two pronuclei zygotes, but no syngamy or cleavage. Three more ICSI attempts were performed, carefully avoiding sperm with obvious anomalies of the connecting piece. Fertilization and cleavage took place in all cycles, and in two of them positive betahCG plasma levels were detected but preclinical abortions ensued. CONCLUSIONS: We propose that the alterations in the head-tail junction and attachment, responsible for the observed sperm phenotype, result from centriolar dysfunctions that cause insufficient sperm aster formation, lack of syngamy and cleavage or defective embryos leading to early abortions.

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

PURPOSE: To analyze the distribution of a tubulins and acetylated alpha 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 > or = 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 > or =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.

Conservative management versus aspiration of functional ovarian cysts before ovarian stimulation for assisted reproduction.

PURPOSE: To compare the efficiency of transvaginal ultrasound-guided functional ovarian cyst aspiration, with conservative management, in the outcome of patients undergoing assisted reproductive technique (ART) (in vitro fertilization or intracytoplasmic sperm injection). These cysts were identified before ovarian stimulation begun and after administration of a midluteal GnRH agonist. METHODS: Fifty nine patients undergoing ART from January 1, 1997 to February 28, 1999, who developed functional ovarian cysts were included. Aspirations of these cysts (n = 14) versus conservative management (observation) (n = 45) were compared. Total number of ovarian follicles developed, number of oocytes retrieved, estradiol levels on the day of human chorionic gonadotropin, fertilization rate, number of good quality embryos transferred, implantation, and clinical pregnancy rate per cycle were evaluated. RESULTS: No statistical differences were observed between the two groups in any of the selected parameters. CONCLUSIONS: Cyst aspiration and conservative management showed similar implantation and pregnancy rates, in patients who develop functional ovarian cysts after pituitary down-regulation following luteal phase gonadotropin-releasing hormone agonist administration. Prospective studies are needed to confirm this trend.

Estudio prospectivo randomizado comparando la via de administración subcutáneas vs. la via intramuscular de depósito de acetato de leuprolide en técnicas de reproducción asistida / Randomized study comparing subcutaneous administration of vial vs. the intramuscular vial of acetate in techniques assited reproduction

Comparar los resultados en técnicas de reproducción asistida (ART) empleando dos vias diferentes de administración de análogos de GnRH (diario vs. depósito9

Estudio prospectivo randomizado comparando la via de administración subcutáneas vs. la via intramuscular de depósito de acetato de leuprolide en técnicas de reproducción asistida / Randomized study comparing subcutaneous administration of vial vs. the intramuscular vial of acetate in techniques assited reproduction

Comparar los resultados en técnicas de reproducción asistida (ART) empleando dos vias diferentes de administración de análogos de GnRH (diario vs. depósito9(AU)

Non-competitive anti-oestrogenic activity of progesterone antagonists in primate models.

We have summarized some of the studies containing basic biological data suggesting potential therapeutic utility of the anti-proliferative activity of antiprogestins on uterine tissues. The non-competitive anti-oestrogenic effects of RU486 were examined using oestradiol-treated ovariectomized monkeys given RU486, progesterone or both. The oestradiol-induced luteinizing hormone surge of control animals was abrogated by progesterone and/or RU486. Secretory transformation by progesterone was inhibited by RU486 co-administration. RU486 alone (1 mg/kg) induced endometrial secretory transformation, but higher doses (5 mg/kg) induced inhibited proliferation and secretory activity. Thus, in the presence of progesterone, RU486 is antagonistic but, in its absence, RU486 exhibits endometrial progestational effects at low doses and an anti-proliferative (anti-oestrogenic) effect at higher doses. These data encourage continued evaluation of RU486 as a potential contraceptive agent acting at the pituitary and/or endometrial level. Our study also demonstrates that after physiological oestradiol replacement therapy, oestradiol receptor concentrations rise dramatically following antiprogestin treatment; this effect was dose-dependent.

Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm.

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.

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).

Effects of ovarian tissue reduction on the menstrual cycle: persistent normalcy after near-total oophorectomy.

These experiments were designed to evaluate whether removal of approximately 95% visible ovarian tissue would interrupt the short- or long-term regulation of cyclic ovarian function. On cycle Days 2 4 (onset of menses = Day 1), the entire left ovary and approximately 90% of the right ovary were removed from three cycling cynomolgus monkeys. After approximately 95% ovariectomy, there was an acute elevation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which lasted 11 +/- 2 days. A midcycle-like gonadotropin surge occurred 20 +/- 3 days following approximately 95% ovariectomy; the next menses occurred 19 +/- 1 days later. Follicular phase patterns of estradiol preceded the midcycle gonadotropin surge, and luteal phase progesterone levels indicated subsequent ovulation. Two of three monkeys resumed normal menstrual cyclicity in the following cycle with follicular phase, luteal phase, and menstrual cycle lengths similar to pretreatment levels. Histological examination of the ovarian remnant removed on Day 21 of the next cycle revealed a morphologically normal corpus luteum and many small follicles. A second group of 6 rhesus monkeys also underwent approximately 95% ovariectomy for long-term evaluation of menstrual cyclicity; typical 28-day menstrual cycle patterns were observed in 4 of the 6 monkeys for 5 mo, with 2 of these 3 animals maintaining regular menstrual cycles for 1 yr. In summary, our data suggest that normal ovarian function, i.e. recruitment, selection, and dominance of the ovulatory follicle, ovulation, and subsequent corpus luteum function, is maintained with only approximately 5% of functional ovarian tissue remaining.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

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.