Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines.

OBJECTIVE: To investigate the use of donated gametes for the production of human embryonic stem cell lines. DESIGN: Basic research study. SETTING: Assisted Reproductive Technology (ART) program at an academic institution. PATIENT(S): Consenting oocyte and sperm donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Oocytes were aspirated from oocyte donors (n = 12) and inseminated with frozen-thawed donor (n = 2) sperm followed by culture of embryos to day 5 or 6 in sequential media. The inner cell masses of expanded blastocysts were isolated using immunosurgery and cultured for 4-11 days on irradiated primary mouse embryonic fibroblasts (PMEFs). Viable cell colonies were passed every 7-10 days onto fresh PMEFs in the presence of leukemia inhibitory factor (0.1 microg/mL) and evaluated for appropriate cell surface markers. RESULT(S): Immunosurgery of 40 blastocysts resulted in the culture of 18 inner cell masses, which have produced three cell lines. One of these cell lines has been shown to stain positive for alkaline phosphatase and stage-specific embryonic antigen (SSEA)-4 and negative for SSEA-1, express telomerase activity, and produce hCG when allowed to differentiate. CONCLUSION(S): These findings demonstrate that the future production of human embryonic stem cell lines for therapeutic use is possible with the use of donated gametes. Many ethical issues were considered before the initiation of this study, and it was our goal to ensure that both oocyte and sperm donors understood the nature and purpose of the research before their participating in the study.

Differential binding of X- and Y-chromosome-bearing human spermatozoa to zona pellucida in vitro.

The sex of human offspring has been associated with the day in the mother's menstrual cycle on which insemination occurs, with male zygotes being formed earlier in the fertile period than female zygotes. Using an in vitro environment designed to mimic the in vivo milieu, we tested the hypothesis that Y-chromosome-bearing spermatozoa survive functionally longer than X-chromosome-bearing spermatozoa, and that this differential functional survival is a contributing factor to the in vivo phenomenon. Donor semen was processed by swim-up and incubated at 37 degrees C in culture medium for 0, 24 and 48 h, with human zona pellucida (hemizona, HZ) being used to select functional spermatozoa. A second set of in vitro storage conditions, 4 degrees C in test-yolk refrigeration buffer, was used to determine whether changing the incubation conditions alters the process. The sex chromosome of the spermatozoa was determined using fluorescent in situ hybridization (FISH). For spermatozoa incubated at 37 degrees C, the percentage of functional (HZ bound) Y-bearing spermatozoa was significantly increased (P < 0.05) at 48 h (55.4 + 2.9%) but not at 0 h (50.5 + 0.7%) or 24 h (52.8 + 3.1%) compared to swim-up spermatozoa (50.6 + 0.3%). No difference in the percentage of functional Y-spermatozoa was observed at any time-point with storage at 4 degrees C in refrigeration buffer. Thus, we demonstrated that significantly more Y-bearing spermatozoa were capable of zona binding than X-bearing spermatozoa at 48 h at 37 degrees C incubation, with an observed Y : X ratio of 1.15 for these zona-bound spermatozoa compared to 1.02 for post-swim-up spermatozoa. We conclude that a differential functional survival appears to exist between X-bearing and Y-bearing spermatozoa, with the latter exhibiting a longer functional survival under in vitro conditions.

Preclinical experience with two selective progesterone receptor modulators on breast and endometrium.

Org 31710 and Org 33628 are two highly selective progesterone receptor modulators (PRMs) with respect to their anti-progestational and anti-glucocorticoid activity. The compounds have been studied both in vitro and in vivo. Org 33628 has approximately four times stronger anti-progestational activity in vitro than does Org 31710, and in rats it is about 15 times more potent in the pregnancy interruption test. Two main indications for the use of PRMs are breast cancer and fertility regulation. The effects of both Org 31710 and Org 33628 were tested in relevant models for these indications. The effects of the two compounds on breast tumor development were assessed and in rats using the DMBA model. Their potency in menses induction was tested in monkeys on a 4-day regimen in the luteal phase, and after a single dose at day 21 of the normal cycle, and under a continuous progestin treatment using desogestrel. The compounds were also tested alone in a continuous low-dose regimen. The effects on follicular development and ovulation were determined by measuring estradiol and progesterone levels. Cycle control was monitored by daily vaginal swabs. In the DMBA model, Org 31710 at oral doses of 0.8, 2.0, and 5.0 mg/kg showed a clear dose-related reduction in tumor load. With the two highest doses, an even lower tumor load was seen after a 3-week treatment period compared to the tumor load at the start of treatment. Org 33628 showed a similar efficacy as Org 31710 at a dose of 2.0 mg/kg. RU 486 after oral treatment was two times less potent in this model than Org 31710 and Org 33628. The efficacy of menses induction using the 4-day regimen is dependent on the time of administration relative to the progesterone peak in the luteal phase. The highest efficacy is achieved in the descending part of the peak, at which a 100% success rate is found with a dose of 1 mg/kg of either Org 31710 or Org 33628. In Cynomolgus monkeys, at a single dose of 15 mg/kg of Org 31710 or Org 33628 in the luteal phase, menses induction was achieved only in 60% of the treatment cycles. Surprisingly menses induction can be achieved with a single dose that is about a ten-times lower when the monkeys are treated continuously with desogestrel. Cycle control is better at low than at high doses of antiprogestin in combination with daily dosing of 4 microg/kg desogestrel. Despite the difference in receptor affinity, no difference between Org 31710 and Org 33628 was found in menses induction. In the continuous low-dose (1 mg/kg) regimen with the PRMs, follicular development occurs normally while ovulation is inhibited. Ovulation is resumed shortly after stopping treatment, and a normal menses occurs after the first progesterone peak. Both compounds may be interesting options for the prevention and treatment of breast cancer and for fertility control.

Incidence of aneuploid spermatozoa from subfertile men: selected with motility versus hemizona-bound.

Spermatozoa-zona pellucida binding selects for human spermatozoa with progressive motility, normal morphology and functional competency. We postulated that this gamete interaction would also act to select against spermatozoa with chromosomal numerical aberrations. Spermatozoa from 41 men participating in the intracytoplasmic sperm injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The hemizona assay was utilized to determine whether zona-bound spermatozoa from these patients have a reduced incidence of aneuploidy compared with those selected by motility only in a standard swim-up procedure. Using multicolour fluorescence in-situ hybridization (FISH) with DNA probes specific for chromosomes 18, X and Y, the disomy rates for chromosomes 18, X, Y and XY were found to be 0.31, 0.27, 0.29 and 0. 14% respectively in the swim-up motile fraction, and 0.31, 0.33, 0. 32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound spermatozoa, the disomy rates for chromosome 18, X, Y and XY were found to be 0.02, 0.15, 0.12 and 0.07% respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up motile fraction or the pellet fraction (P < 0.0001). The incidence of chromosome 18 aneuploidy, including both chromosome 18 disomy and nullisomy, in the swim-up motile fractions was significantly increased in patients with an abnormal or borderline hemizona index compared with those with a normal hemizona index (P < 0.05). We also found that a high incidence of sperm aneuploidy was associated to a certain extent with low fertilization rate, and with failure to achieve pregnancy through ICSI. This study suggests that the human zona pellucida has the capacity to select against aneuploid spermatozoa by an as yet undetermined mechanism.

Variations in plasma levels of substance P and effects of a specific substance P antagonist of the NK(1) receptor on preovulatory LH and FSH surges and progesterone secretion in the cycling cynomolgus monkey.

These studies investigated the role of substance P (SP) in the regulation of the hypothalamic-pituitary-ovarian axis in cynomolgus monkeys with normal menstrual cycles. Plasma concentrations of SP were determined in blood samples taken every morning in normally menstruating cynomolgus monkeys throughout the menstrual cycle. There was a significant decreasing linear trend of SP during the follicular phase (cycle day -13 to day 0) and a significant inverse relationship between SP plasma values and plasma 17beta-estradiol (E(2)) values from day -13 to day 0 of the adjusted cycle. Correspondingly, SP area under the curve was significantly greater during the follicular phase than the luteal phase. In a second experiment, plasma concentrations of E(2), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone and length of cycles were measured after five daily intragastric administrations (10 mg/kg) of an NK(1) receptor (SP receptor) antagonist (RPR 100893; 10 mg/kg) initiated after serum E(2) concentrations had exceeded 125 pg/ml. There was a statistically significant reduction in the amplitude (41% of control) and the area under the curve (37% of control) of the preovulatory LH surge. In addition, there was a reduction of the duration of the LH surge (3 +/- 0.1 days in controls vs. 2.1 +/- 0.2 days in treated animals). The present results show for the first time that there are significant variations in plasma levels of SP, with a strong negative correlation with serum levels of E(2) during the follicular phase of the cynomolgus monkey, and that endogenous SP has a potentiating role in the interactive hypothalamo-anterior-pituitary mechanisms which lead to the preovulatory LH and FSH surges during the menstrual cycle in the monkey.

Disparate actions of mifepristone (RU 486) on glands and stroma in the primate endometrium.

Besides being an antiprogestin, mifepristone (RU 486) was recently shown to antagonize oestrogen-dependent growth in the endometrium. To explore the molecular mechanisms for this phenomenon, we investigated whether or not the morphological effects of mifepristone are mediated by the progesterone receptor (PR) and whether mifepristone has disparate effects on the glandular epithelium and stroma. Six groups of hypogonadal, oestrogen-primed cynomolgus monkeys were treated for 2 weeks with: vehicle only (group I); mifepristone (group II); mifepristone plus progesterone at 0.2 mg/kg (group III), 1.0 mg/kg (group IV) or 5.0 mg/kg (group V); and progesterone only (5.0 mg/kg) (group VI). Histomorphological evaluation showed strikingly compacted stroma in the mifepristone-exposed endometria (group II), which was partially reversible by additional progesterone treatment (groups III-V). Glandular proliferation (pseudostratification, glandular mitoses) in mifepristone-treated monkeys was not significantly different from that in vehicle (oestradiol)-treated monkeys, but was inhibited by progesterone-only treatment. Cells containing vacuoles were scarce in the mifepristone-exposed endometrium, but detected frequently in progesterone-exposed endometria, indicating the strong antisecretory effect of mifepristone on glands. We conclude that oestrogen-dependent oedema in the stroma is antagonized by mifepristone. The reversal of this effect by progesterone suggests a PR-mediated mechanism. In glands, mifepristone is antiprogestogenic, but not antioestrogenic. Thus, stromal cells may be the target of antiprogestin-induced inhibition of oedema and endometrial growth.

Screening for antiproliferative actions of mifepristone. Differential endometrial responses of primates versus rats.

This laboratory has previously shown the capability of the antiprogestin, mifepristone, to noncompetitively inhibit estrogen-induced endometrial proliferation in nonhuman primates. In the following study, use of the rat uterine weight bioassay was compared against a primate (Macaca fascicularis) uterine bioassay to identify the noncompetitive/antiproliferative effects of mifepristone. These uterine bioassays were contrasted for reasons of identifying a comparative laboratory rodent model that could substitute for the need to use primate models in the screening of potential antiprogestins, thereby saving time, cost, and primate resources. Results of the primate experiment showed that mifepristone decreased endometrial proliferation in a dose-dependent manner; importantly, this decrease occurred in the presence of sustained physiologic serum 17 beta-estradiol (E2) levels. However, in the rat model, results showed that mifepristone altered uterine wet weight and blotted weight values only in those animals receiving pharmacological doses of E2 (p < 0.05). Based on the results summarized herein, use of this rat uterine weight bioassay as a substitute for primate models is not recommended for screening and identification of "interesting" antiprogestins. Apparently, the endometrial noncompetitive antiestrogenic/antiproliferative effects of mifepristone, observed repeatedly in these laboratory primates, do not operate in the rat uterine tissue.

Chronic antiprogestin therapy produces a stable atrophic endometrium with decreased fibroblast growth factor: a 1-year primate study on contraception and amenorrhea.

OBJECTIVE: To describe the efficacy of mifepristone in the prevention of menstrual bleeding and ovulation, with similar observations in comparison groups. DESIGN: Prospective experimental study. Thirty-two cynomolgus monkeys were divided equally into four treatment groups (n = 8). Treatment lasted for 1 year. INTERVENTION(S): Group I received GnRH-agonist (GnRH-a) and in-sequence mifepristone, group II received mifepristone only, group III received GnRH-a only, and group IV received vehicle control. MAIN OUTCOME MEASURE(S): Serum estradiol and progesterone, menstrual bleeding, endometrial thickness, and endometrial expression of basic fibroblast growth factor (bFGF) as determined by immunohistochemistry. RESULT(S): Weekly progesterone determinations showed that mifepristone-treated monkeys seldom ovulated (6 ovulations in 8 years), compared with the controls (100 ovulations in 8 years), while maintaining early to midfollicular levels of circulating serum estradiol. The GnRH-a-only group also rarely ovulated, but was chronically and severely hypoestrogenic. The mifepristone-only group showed scant menstrual bleeding (5 days in 8 years) as compared with the menstrual frequency in control animals (422 days in 8 years). Endometrial proliferation, as determined by biopsy, was similarly minimal for both the GnRH-a and mifepristone groups, and statistically less than in control monkeys. Both the mifepristone and GnRH-a treatments suppressed endometrial gland expression of the angiogenesis polypeptide bFGF. CONCLUSION(S): Chronic mifepristone induced anovulation along with virtual amenorrhea, which suggests the worth of this novel hormonal contraceptive.

Vascular endothelial growth factor in primate endometrium is regulated by oestrogen-receptor and progesterone-receptor ligands in vivo.

We investigated hormonal regulation of endometrial angiogenesis in menstruating primates. This study was designed to demonstrate: (i) that cell-specific vascular endothelial growth factor (VEGF) production and expression in monkey endometrium are regulated by steroid receptor ligands; and (ii) mifepristone (RU 486) alters VEGF production even in the absence of a progestin agonist. Endometrial VEGF production was compared by computer-assisted immunohistochemical analysis during induced hypoestrogenism and after oestradiol, progestin, or antiprogestin (mifepristone) treatment. VEGF gene expression was estimated by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in endometrial samples from castrate cynomolgus monkeys, from intact monkeys in the luteal phase, and from monkeys treated for 20 days with levonorgestrel (LNG) or mifepristone. VEGF staining intensities in glandular epithelium and VEGF mRNA expression were highest in hypoestrogenic monkeys. Progestin treatment induced intense VEGF staining in the stroma. Gene expression of VEGF-189, but not other isoforms, was higher in progesterone- and progestin (LNG)-exposed endometria compared to mifepristone-exposed endometria or endometria from anovulatory cycles (P < 0.04). Mifepristone abolished VEGF staining in glandular epithelium almost completely. We conclude that VEGF protein and VEGF mRNA expression levels in primate endometrium depend on the steroidal milieu. Anti-angiogenic effects of mifepristone via suppression of VEGF production might represent a mechanism for its quelling effects on endometrium.

Hemizona assay: evaluation of fertility potential in patients with vasectomy reversal.

Approximately 40 to 60% of men who undergo a successful vasovasostomy have "functional failure" or failure to conceive even though active sperm are present. Factors of this functional failure include sperm abnormalities (oligoasthenoteratospermia) and antisperm antibodies. Nine male patients 32 to 43 years of age who underwent vasovasostomy were included in the study group. These patients demonstrated ductal patency 3 to 6 months after surgery. After attempts at fertility with their spouses failed, the couples underwent urological and gynecological evaluation. Semen parameters were recorded and further evaluation was performed using immobilizing and agglutination antibodies as well as direct immunobead test and the hemizona assay (HZA). Semen parameters presented varying levels of sperm concentration, percent motility, and morphology (strict). Antisperm antibodies were present in 4 of the 9 patients. Three of 4 patients with antibodies and 4 of 5 patients without antibodies benefitted from hemizona assay results in that it either supported a desired therapy or gave objective data that would dictate more aggressive therapy. Six men had a hemizona index of > 35%, predictive of adequate zona bind capability. Using these individual situations combined with gynecologic findings, recommendations are made as to identifying realistic options and therapeutic recommendations.

Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the nonhuman primate (Macaca fascicularis) epididymis.

The 5alpha-reduced metabolites of testosterone, including dihydrotestosterone, are considered the primary regulators of epididymal function. Two genes encode two 5alpha-reductase isozymes. We examined 5alpha-reductase type 1 and type 2 mRNA tissue distribution and relative abundance in cynomolgus monkey (Macaca fascicularis) testicular and epididymal tissues using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA extracted from monkey tissues including the testis (T) and the proximal caput (PCp), the caput (Cp), the midcorpus (Co), and the distal cauda (Cd) epididymis was reverse transcribed to produce cDNAs. 5alpha-reductase type 1 and 2 cDNAs were subsequently coamplified with the housekeeping gene, cyclophilin, in a PCR spiked with 33P-dCTP. Relative abundance was reported as the cpm ratios of type 1 or type 2/cyclophilin mRNA. Semiquantitative RT-PCR results indicated that type 1 mRNA was most abundant in the testis (0.48 +/- 0.06) and significantly decreased distally along the monkey epididymis (PCp: 0.29 +/- 0.04; Cp: 0.29 +/- 0.04; Co: 0.21 +/- 0.03; Cd: 0.07 +/- 0.01) (P < 0.001). Type 2 mRNA was undetectable in the testis but was present throughout the epididymis at uniform levels (PCp: 1.6 +/- 0.2; Cp: 1.4 +/- 0.3; Co: 1.6 +/- 0.2; Cd: 1.5 +/- 0.2). These data demonstrate that 5alpha-reductase type 1 mRNA is differentially expressed but of low abundance along the nonhuman primate epididymis, whereas 5alpha-reductase type 2 gene expression is uniform.

Endometrial effects of RU486 in primates--antiproliferative action despite signs of estrogen action and increased cyclin-B expression.

Continuous antiprogestin administration to hormone replaced, castrate monkeys inhibits estrogen-induced endometrial proliferation through mechanisms which remains unclear. To elucidate the molecular mechanisms of RU486-induced endometrial suppression, we treated six intact female cynomolgus monkeys on cycle days 2-22 sequentially with placebo, RU486 (1 mg/kg/day) and levonorgestrel (LNG) (2 microg/kg/day) intramuscularly (i.m.), with uterine wedge sections and endometrial biopsies collected on day 22 of each cycle. The uterine sections were evaluated for morphology, mitosis and proliferating cell nuclear antigen (PCNA) immunohistochemistry. Changes in the mRNA levels of ER, PR, cyclin-B and tumour suppressor gene p21 were assessed using co-amplification with beta-actin by reverse transcriptase-polymerase chain reaction (RT-PCR). Administration of RU486 uniformly resulted in characteristic suppression of endometrium with few mitosis, dense stroma and simple glands, whereas the effects of LNG were less uniform. Following RU486 administration, the levels of endometrial ER and PR mRNA were comparable to proliferative phase endometrium, and significantly higher than those seen in the secretory endometrium, indicating that some of the biological actions of E2 were not inhibited during RU486 treatment. Despite scarce mitosis, PCNA was readily detectable in all samples. Curiously, in comparison to secretory phase controls, the levels of cyclin-B, but not p21, mRNA were markedly increased following RU486. The effects of LNG on the levels of these mRNA species varied, with mean levels falling between those of the secretory phase controls, and RU486-treated specimens. The increase in cyclin-B mRNA and lack of mitosis suggests that anti-proliferative actions of RU486 in the primate endometrium might be associated with a cell-cycle block at the G2-M interphase. Whether mechanisms similar to these are associated with the beneficial clinical effects of RU486 seen in the treatment of various hormone dependent maladies remains to be determined.

Ectopic growth of endometrium depends on its structural integrity and proteolytic activity in the cynomolgus monkey (Macaca fascicularis) model of endometriosis.

OBJECTIVE: To identify factors influencing the development of endometrial autografts in a monkey model of endometriosis. DESIGN: Prospective, comparative study. SETTING: Animal research unit. SUBJECTS: Thirty regularly cycling cynomolgus monkeys in three groups of 10 each. INTERVENTIONS: Endometrium was minced and spilled into the cul-de-sac in group 1. In group 2, the tissue additionally was digested enzymatically. In group 3, the tissue was incubated with a protease inhibitor. MAIN OUTCOME MEASURES: Staging laparotomies after 3 weeks and 3 months. RESULTS: In groups 1, 2, and 3, moderate or severe disease was seen in eight, two, and four monkeys, respectively, after 3 weeks and in eight, three, and two monkeys, respectively, at 3 months. CONCLUSIONS: An intact structure leads to ectopic implantation of endometrial fragments in most cases. Conversely, enzymatic digestion of endometrial fragments and treatment with proteinase inhibitor impair ectopic growth. Intrinsic endometrial factors that influence extracellular matrix remodeling may play a role in the pathogenesis of human endometriosis.

Role of hypoestrogenism or sex steroid antagonism in adhesion formation after myometrial surgery in primates.

OBJECTIVE: To determine the contribution of estrogen in the development of pelvic adhesions during myometrial surgery. DESIGN: A randomized, prospective study in the nonhuman primate. SETTING: A primate colony, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. INTERVENTIONS: All primates were assigned prospectively to one of three treatment groups: [1] GnRH analogue (GnRH-a), [2] mifepristone, or [3] vehicle control. After 3 months of treatment, a standard uterine fundal hysterotomy, for full thickness endometrial biopsy, was performed at the time of exploratory laparotomy, with subsequent scoring of utero-omental adhesions to the hysterotomy site at a future staging procedure based upon adhesion area, vascularity, and tenacity. Serum was drawn on the day of surgery for E2 determination. Endometrial height, from the surface interface between the endometrium and myometrium, was used as a bioassay of estrogen activity. RESULTS: The hypoestrogenic (GnRH-a) group and the mifepristone group had significantly fewer utero-omental adhesions compared with the normally cycling control monkeys as measured by a lower adhesion score. Similarly, the endometrial thickness was significantly reduced in the GnRH-a and mifepristone groups (one-third) compared with the cycling controls, demonstrating the effects of either hypoestrogenism or noncompetitive estrogen antagonism. Serum E2 on the day of surgery was predictive of the postoperative adhesion score by both a regression analysis and analysis of covariance. CONCLUSIONS: The actions of E2 seem to have a dramatic effect on the formation of pelvic adhesions after myometrial surgery.

Non-competitive anti-oestrogenic actions of progesterone antagonists in primate endometrium: enhancement of oestrogen and progesterone receptors with blockade of post-receptor proliferative mechanisms.

Previous studies have shown that the progesterone antagonists (antiprogestins) inhibit oestrogen-dependent endometrial proliferation in ovariectomized monkeys, without having affinity to the oestrogen receptor (ER). This study was designed to investigate the effect of the antiprogestins mifepristone (RU 486) and onapristone (ZK 98,299), on the concentration of ER and progesterone receptor (PR) in the endometrium of long-term ovariectomized cynomolgus monkeys (Macaca fascicularis). In untreated monkeys, tissue preparations bound in total 228 +/- 68 pmol [3H]-oestradiol/g protein (ER), and 119 +/- 42 pmol [3H]-R5020/g protein (PR). These values were not significantly different from the total binding capacities of tissues from monkeys treated with RU 486 alone or primates treated with oestradiol plus progesterone. Treatment with oestradiol alone almost doubled the ER and PR concentrations. Combined treatment with oestradiol and RU 486 enhanced the ER and PR concentrations in a dose-dependent manner: 1 mg/kg body weight (bw) RU 486/kg increased both ER and PR contents about 3-fold. The dose of 5mg/kg bw RU 486 or onapristone increased the ER and PR concentrations almost 6- and 5-fold respectively, compared with the oestradiol-treated controls. Our results demonstrate that RU 486 and onapristone increased the endometrial ER and PR concentrations far beyond the physiological level in ovariectomized, oestradiol-treated monkeys. Whether the over-expression of ER and PR in the presence of antiprogestins is causally related to the antiproliferative impact of antiprogestins in the endometrium (non-competitive anti-oestrogenic effects) or is an independent action in unknown.

Antiprogestin and/or gonadotropin-releasing hormone agonist for endometriosis treatment and bone maintenance: a 1-year primate study.

The fact that RU 486 curtailed estrogen-induced endometrial proliferation in primates and relieved pelvic pain in women with endometriosis is the reason for continuing research on antiprogestins. Thirty-two adult female cynomolgus monkeys demonstrating menstrual regularity had surgery for the induction of endometriosis. After lesion staging, four treatment groups (n = 8), each of 1-yr duration, were made. Group I received combination/sequential therapy with depot GnRH agonist (GnRH-a) for 3 months, followed by weekly RU 486 for 9 months. Group II received weekly RU 486, group III received monthly GnRH-a, and group IV served as a vehicle control. A staging laparotomy was performed every 3 months to assess the area of peritoneal endometriosis (square centimeters) and the thickness of in situ endometrium. Bone density was measured serially by dual x-ray absorptiometry. Serum was collection weekly. Mean (+/- SE) serum estradiol levels were lower after GnRH-a (77.1 +/- 2.6 pmol/L) than after RU 486 (231 +/- 12 pmol/L) treatment and lower than those in untreated cycling controls (231 +/- 13 pmol/L). GnRH-a produced significant atrophy of endometriotic plaques within 3 months of therapy; this lesion reduction was sustained with RU 486. Both GnRH-a and RU 486 alone produced profound thinning of ectopic and eutopic endometrium throughout 1 yr of continuous therapy. Bone density decreased significantly after 6 months of GnRH-a alone (P < 0.05), without significant changes in the other groups. After RU 486 treatment, there were no significant changes in testosterone, androstenedione, sex hormone-binding globulin, or cortisol. Like GnRH-a, long term antiprogestin therapy produced a reduction in the volume of pelvic endometriotic lesions as well as atrophy of in situ endometrium; however, RU 486 allowed maintenance of tonic ovarian estradiol secretion, suggesting that efficacious endometriosis therapy can be sustained long term without the sequelae of hypoestrogenism, specifically bone density loss.

Effects of caffeine and dbcAMP on zona pellucida penetration by epididymal spermatozoa of cynomolgus monkeys (Macaca fascicularis).

Spermatozoa mature during epididymal transit, acquiring the abilities to swim progressively, fertilize oocytes, and produce viable offspring. In this study, we investigate the capacity of spermatozoa retrieved from the midcorpus and distal cauda regions of the epididymis of the cynomolgus monkey to penetrate homologous zona pellucida. Successful in vitro fertilization by ejaculated macaque sperm is dependent upon the addition of caffeine and dbcAMP. Therefore, the effect of these cyclic nucleotide mediators was also examined in this study. Results of sperm motion analysis indicate no difference in baseline values (without stimulators) for any motion parameter. With the addition of caffeine and dbcAMP, curvilinear velocity significantly increased only for the distal cauda sperm (P = 0.05). Amplitude of the lateral head displacement was significantly increased for distal cauda sperm (P < 0.01); although elevated above baseline, the increase observed after activation by corpus sperm was significantly lower than that achieved by cauda sperm (P < 0.05). The addition of caffeine and dbcAMP was an absolute requirement for zona penetration by both midcorpus and distal cauda sperm. With activation, zona penetration was significantly decreased for corpus sperm compared to cauda sperm (P < 0.001). These results suggest that cynomolgus monkey sperm reaching the midcorpus region of the epididymis have not completed all of the maturational changes requisite for successful fertilization; this immaturity is evidenced by decreased sperm motion and by impedance at the level of zona penetration.

Multiple transcription start sites for the GnRH gene in rhesus and cynomolgus monkeys: a non-human primate model for studying GnRH gene regulation.

In humans, transcription of the gonadotropin-releasing hormone (GnRH) gene can be initiated at two transcription start sites to produce different GnRH mRNAs. The upstream transcription start site is used only in reproductive tissues and tumors. To determine if a similar pattern of GnRH gene expression exists in non-human primates, we cloned GnRH cDNA from rhesus monkey hypothalamic RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and the 5' flanking region of the monkey GnRH gene by PCR. A 96% similarity between monkey and human GnRH cDNA was found with 94% similarity in the upstream promoter region. An upstream transcriptional start site, was identified in cynomolgus monkey testicular mRNA, 504 base pairs upstream from the hypothalamic site, which was different from that identified in the human GnRH gene. Various cynomolgus monkey reproductive tissues were found to utilize this upstream transcriptional start site. In contrast, no evidence was found for the use of upstream transcriptional start sites in rat testis or placenta, suggesting that the reproductive tissue specificity of the upstream transcription start site may be a primate specific feature.

Antiproliferative effects of low-dose micronized progesterone.

OBJECTIVE: To study the pharmacodynamic effects of oral micronized P on endometrial maturation. DESIGN: This was a controlled, open, parallel group, pilot study. SETTING: The experiment was performed in an outpatient academic clinical research unit. PATIENTS: Twelve healthy, P-challenged, estrogen-primed, postmenopausal women participated in the study. INTERVENTIONS: Patients were given 300 mg micronized P daily (8:00 A.M.) or twice (8:00 A.M. and 4:00 P.M.) daily from study days 1 through 14 after estrogen priming for 30 days. Blood samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours after the 8:00 A.M. dose on study day 1 and 14 and again at 8:00 and 9:30 A.M. on days 3 and 5 fasting, days 7 and 9 after a fatty meal, and day 11 after a high fiber meal. Endometrial biopsies were taken on day 1 and 14. MAIN OUTCOME MEASURES: Progesterone concentrations were measured. Endometrial biopsies were studied for effects on histology, glycogen content of glands, ribosomal RNA, and nuclear estrogen receptors in glands, surface epithelium, and stroma. RESULTS: Day 1 and 14 P kinetics were similar for 8 hours. Dose-dependent increases in glandular glycogen, decrease in ribosomal RNA, and decrease in nuclear estrogen receptors were demonstrated. CONCLUSIONS: Oral micronized P can induce antiproliferative changes in the human endometrium at doses lower than those required for transformation of the endometrium to a full secretory state.

The effect of coculture on the postfertilization development of in vitro-matured monkey oocytes.

OBJECTIVE: To determine if the developmental potential of embryos resulting from in vivo- and in vitro-matured monkey oocytes could be increased through the use of a coculture system. DESIGN: Randomized prospective comparison of embryos resulting from either in vitro- or in vivo-matured oocytes cocultured with Vero cells or cultured in medium alone (control). SETTING: Basic research laboratory. MAIN OUTCOME MEASURES: In vitro embryo development to the blastocyst stage and blastocyst hatching. RESULTS: No significant difference in development was noted between coculture and control groups with embryos resulting from in vivo-matured oocytes. However, coculture was found to improve significantly the development of monkey embryos resulting from in vitro-matured oocytes. CONCLUSIONS: These results demonstrate that primate embryos resulting from in vitro-matured and in vitro-fertilized oocytes differ in their culture requirement when compared with embryos resulting from in vivo-matured oocytes.