Changes in adenylyl cyclase activity of the human and nonhuman primate corpus luteum during the menstrual cycle and pregnancy.

Adenylyl cyclase (AC) activity in membrane particles of corpora lutea (CL) from humans and cynomolgus monkeys was examined at various stages of the menstrual cycle and pregnancy. AC activity was monitored by the conversion of [alpha-32P]ATP into [32P]cAMP under basal conditions and in the presence of several activators: NaF (10 mmol/L) plus forskolin (100 mumol/L); hCG (10 micrograms/mL); guanyl 5'-yl-imidodiphosphate [GMP-P(NH)P; 100 mumol/L]; and hCG (10 micrograms/ml) plus GMP-P(NH)P (100 mumol/L). The groups of human CL were midluteal (n = 10), late luteal (n = 4), following cycle (old CL; n = 5), and early pregnancy (6-11 weeks; n = 10). The groups of monkey CL were early luteal (n = 4), midluteal (n = 5), and pregnancy at term (n = 3). Luteal AC activity changed significantly during the menstrual cycle. In newly (less than 48 h after ovulation) formed CL, the enzyme was unresponsive to hCG, and total AC activity, as determined by NaF plus forskolin, averaged 86.5 +/- 28.9 (+/- SE) pmol cAMP/min.mg protein. As the CL developed, AC activity increased. Thus, in the midluteal phase, maximal hCG responsiveness in the presence of guanine nucleotide was 125 +/- 27 and 232 +/- 15 pmol/min.mg in human and monkey CL, respectively. No hCG responsiveness was detected in the late luteal phase or in the old CL. Maximal AC activity was also high in the midluteal phase (382 +/- 56 and 256 +/- 28 pmol/min.mg in human and monkey CL, respectively); the activity remained fairly high during the late luteal phase and then declined to less than 100 pmol/min.mg in the follicular phase of the next cycle. During early pregnancy, luteal AC was unresponsive to hCG stimulation, yet basal levels, maximal activity, and the characteristics of stimulation by nonhormonal activators were similar, if not identical, to those at the midluteal phase of the menstrual cycle. At term pregnancy, the enzyme remained unresponsive to hCG. However, basal activity and stimulation by NaF and forskolin were remarkably elevated, being between 2- and 7-fold higher than corresponding stimulations in the midluteal phase. We conclude that 1) AC activity in human luteal membranes is highly dependent on hormonal changes and functional state of the ovary, 2) the activity of luteal AC is similar in the CL of humans and cynomolgus monkeys, and 3) the AC system in the primate CL is functionally active during and at the end of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Failure of indomethacin to block the estrogen-induced luteinizing hormone surge in the ovariectomized rhesus monkey.

Prostaglandins have been shown to be stimulatory to LH release in several species, although it is not known, in the primate, whether this effect is mediated through the hypothalamic-pituitary axis or by an indirect effect mediated through the ovary. In the present experiments, indomethacin, an inhibitor of prostaglandin synthesis, did not significantly alter basal LH levels or the positive and negative feedback effects of estrogen on LH release in the ovariectomized rhesus monkey. Therefore, it appears that the previously demonstrated effect of indomethacin on LH release in the intact monkey was mediated through ovarian mechanisms.

Suppression of postovulatory gonadotropin levels does not affect corpus luteum function in rhesus monkeys.

The exact mechanism(s) of control of corpus luteum function remains unknown. To address the question whether luteinizing hormone (LH) plays an important role, the following experiments were performed in cycling rhesus monkeys. Experiment 1: 10 monkeys were followed for 2 consecutive cycles and ovulation data determined using serum estradiol (E2) measurements and serial laparoscopies. In the second cycle, starting on day 1 after ovulation, the animals received daily administration of a potent inhibitory LHRH analog, [N-Ac-D-Trp1,3,D-p-Cl-Phe1,D-Phe6,D-Ala10]-LH-RH (N = 5) or vehicle (N = 5) until the onset of vaginal bleeding. Serum FSH and LH concentrations declined significantly in animals treated with the antagonist. However, E2 and progesterone (P) levels were not different from vehicle-treated monkeys. Luteal phase length in both groups was unchanged from the control cycle. Experiment 2: the same protocol was followed, adding the administration of hCG in increasing dosages from days 6-10 after ovulation (Day 6 = 30 IU, Day 7 = 60 IU, Day 8 = 90 IU, Day 9 = 180 IU, Day 10 = 360 IU). The increase in serum P concentration was the same for both groups. These results suggest that significant suppression on gonadotropin levels after ovulation does not affect corpus luteum function and that the inhibitory analog does not have a direct effect on the ovary.

Suppression of the positive feedback of estradiol benzoate on gonadotropin secretion by an inhibitory analog of luteinizing hormone-releasing hormone (LRH) in oophorectomized rhesus monkeys: evidence for a necessary synergism between LRH and estrogens.

Estrogens have a biphasic effect on gonadotropin (FSH and LH) secretion in human and nonhuman primates. Recent studies suggest that estrogens induce negative as well as positive feedback on gonadotropin secretion by a direct action at the pituitary gland. To further investigate the mechanism by which estrogens modulate gonadotropin secretion, the following experiments were performed. Oophorectomized rhesus monkeys were injected with estradiol benzoate (EB) at a dose of 40 micrograms/kg sc at zero time (controls). Three groups of animals received, additionally, an inhibitory analog of LRH, [Ac-D-p-Cl-Phe1,2, D-Trp3,D-Arg6,D-Ala10]LRH (200 micrograms, sc, daily) at 1) -48 to 48 h, 2) 0-48 h, and 3) 24-48 h from EB treatment, respectively. Blood was collected at -72, -48, -24, and 0 h, every 6 h from 6-84 h, and 96 and 116 h after EB; FSH and LH levels were measured by RIA. Control animals had a 70-80% decrease in baseline FSH and LH levels during the first 24 h after EB injection, followed by a sharp rise to 60-80% above basal levels 42-48 h post-EB. In contrast, animals that received the inhibitory analog of LRH in different regimens had more pronounced and prolonged negative feedback of EB on gonadotropin concentrations. The positive feedback release of FSH and LH was significantly diminished or delayed compared to control values. The results of this study indicate that this inhibitory analog of LRH potentiates the negative feedback and suppresses the positive feedback of EB on gonadotropins, suggesting that estrogens induce the secretion of FSH and LH through a mechanism that involves the action of LRH at the gonadotroph.

Effects of hCG on prostaglandin synthesis and function of corpus luteum.

The effect of intramuscular injections of human chorionic gonadotropin (hCG) on subsequent in vitro prostaglandin (PG) production by the corpus luteum was studied in the rhesus monkey. Four monkeys received increasing doses of hCG from days 6 to 10 after ovulation. On day 11, laparotomy and luteectomy were performed. Five untreated animals served as controls. The production of prostaglandins F (PGF) and E (PGE) by the corpus luteum of the animals treated wit hCG was significantly lower than that of the controls (P less than .01). After hCG treatment, the decrease in PGF production was greater than that of PGE, resulting in a lower ratio of PGF:PGE production than in the controls (P less than .01). Histologic evaluation of the corpora lutea revealed regressive changes in the control group, whereas signs of active secretion were observed in the hCG-treated group. Progesterone concentrations in peripheral blood of the hCG-treated group were approximately fourfold higher than in the controls, and the hCG-treated group did not have a postmidluteal phase decrease. These results suggest that the ratio of production of PGF to PGE by the corpus luteum of the rhesus monkey may play a role in the control of its lifespan and steroidogenic capacity.

Can severe male factor infertility be treated without micromanipulation?

OBJECTIVE: To present data on IVF in severe male factor infertility using modified IVF laboratory methods and to compare them with the reported data of gamete micromanipulation. DESIGN: Retrospective, not randomized. SETTING: University of California, Irvine, Center for Reproductive Health. PATIENTS: Seventy-one patients with severe oligoasthenozoospermia defined as total motile count in the pretreatment sample of < 5 x 10(6). Two groups were identified, group I with total motile count between 1.5 and 5 x 10(6) and group II with total motile count < 1.5 x 10(6). INTERVENTIONS: Treatment of the semen samples with mini-Percoll and the modification of standard IVF techniques. These modifications include insemination with larger numbers of sperm, the use of culture tubes for insemination of oocytes, and pooling oocytes together in one or more culture tubes. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates (PRs). Additionally, the number of patients achieving cryopreservation and patients with previous failure to fertilize are specified. RESULTS: Eighty percent of the patients achieved fertilization with an overall rate of 38% per oocyte. Fifty-three ETs were performed and 23 clinical pregnancies (43% per transfer and 35% per cycle) were achieved. Fifty percent of the patients had excess embryos for cryopreservation. In group I the fertilization rate was 54% with a PR of 56% per transfer and 48% per cycle. In group II the fertilization rate was 25% with a PR per transfer of 32% and 24% per cycle. Of 28 patients who had previous failure of fertilization, 25 fertilized and 8 pregnancies were established. CONCLUSIONS: These data demonstrate that by the modification of standard laboratory methods for IVF, monospermic fertilization, cleavage, and a high clinical PR can be achieved in cases of severe male factor infertility without having to resort to micromanipulation.

The importance of egg and sperm factors in predicting the likelihood of pregnancy from gamete intrafallopian transfer.

The relative contribution of factors affecting the probability of pregnancy from gamete intrafallopian transfer was estimated using data from 218 cycles performed in San Antonio, Texas, and Orange, California. Mature eggs, all of which contained an expanded cumulus, were subclassified according to the corona radiata; those with a starburst corona were considered the most fully mature. Sixty-two clinical pregnancies were established, representing a pregnancy rate of 28.4%. Excluding 49 cases of male factor infertility, the pregnancy rate was 34.9%. Pregnancy rates were not significantly different according to age, cause of female factor infertility, parity, duration of infertility, stimulation protocol, or estradiol level. The only variables having a strong association with pregnancy were sperm and egg parameters. Sperm motility and morphology were statistically more important than sperm density. Based on odds ratios calculated from multiple logistic analysis, pregnancy was 3.80 times more likely if three or more fully mature eggs were transferred, 0.34 times as likely if motility were less than or equal to 30%, and 0.22 times as likely if normal morphology was less than or equal to 50%, controlling for other egg and sperm factors. If all egg and sperm factors in a given cycle were unfavorable, the probability of pregnancy estimated by the logistic model was 4%, whereas if all were favorable the probability of pregnancy in that cycle was 59%.

Successful fertilization, pregnancy, and birth using epididymal sperm frozen 24 hours after conventional oocyte insemination.

OBJECTIVE: To assess if epididymal sperm cryopreserved 24 hours after exposure to oocytes in conventional IVF can be successfully used for intracytoplasmic sperm injection (ICSI) in a subsequent cycle. DESIGN: Case report. SETTING: University of California, Irvine, Center for Reproductive Health. PATIENTS: Two men with obstructive azoospermia requiring microsurgical epididymal sperm aspiration, IVF, and ICSI. INTERVENTIONS: Freezing of epididymal sperm 24 hours after egg exposure in conventional IVF and subsequent use for assisted fertilization in a new cycle. MAIN OUTCOME MEASURE: Frozen-thawed epididymal sperm survivability and maintenance of fertilization and pregnancy capacity. RESULTS: At the time of sperm aspiration procedure (cycle I) a total of 30 oocytes were available for insemination. Of these, 15 were used for conventional IVF resulting in 2 embryos (13%) and 15 were used for ICSI, resulting in 3 embryos (20%). Sperm was cryopreserved 24 hours after conventional IVF and thawed 6 months later in a new cycle. Upon thawing, sperm were still found to be motile and at this time (cycle II) only assisted fertilization was used. Of 27 oocytes injected, 12 (44%) produced normal, cleaving embryos. One singleton pregnancy with the birth of a healthy infant girl was achieved after the tubal transfer of 5 embryos. CONCLUSION: The birth of a normal, healthy infant girl with epididymal sperm frozen 24 hours after exposure to oocytes in conventional IVF emphasizes the value of freezing any aliquot of epididymal sperm, even if the motility is very low, to avoid additional surgery in the male. From a basic science standpoint, this observation may renew interest in the study of sperm cryopreservation after occurrence of acrosome reaction and hyperactivation.

Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model.

OBJECTIVES: To use the protein-free medium blastocyst antihatching model to characterize physiological events that mediate hatching. DESIGN: In a series of four prospective experiments, our aims were to [1] test the efficacy of the antihatching model and assisted hatching; [2] exam the influence of initial in vivo developmental stage and late serum supplementation on hatching inhibition; [3] discount the role of zona hardness and physical expansion directly affecting hatching; and [4] provide evidence that the trophectoderm is directly responsible for secreting a zona lysin. SETTING: University-based research laboratory. RESULTS: Culturing two- to eight-cell mouse embryos in serum-free human tubal fluid (HTF) medium significantly reduced hatching levels to < or = 2%, however, hatching increased to 10.7% when initially culturing morula-stage embryos. Hatching was effectively rescued to control levels when embryos were placed in HTF with serum at the early blastocyst stage. There was no difference in blastocyst total cell numbers or zona pellucida digestion intervals between culture treatments. Finally, we showed that trophectodermal vesicles, devoid of inner cell mass, are capable of hatching under control conditions. CONCLUSIONS: The primary mechanism of blastocyst hatching is not physical expansion and abnormal zona hardness is not responsible for hatching inhibition. Certain extracellular precursors found in serum (e.g., amino acids) are required in culture medium upon cellular determination of trophectoderm (i.e., morula to blastocyst stage) to facilitate the intrinsic secretion of an undefined hatching factor.

Platelets bind to rhesus monkey corpora lutea and stimulate its prostaglandin synthesis.

The in vitro production of different prostaglandins (PGs) was studied in rhesus monkey corpora lutea incubated with labeled arachidonic acid. The percentage of conversion of the arachidonic acid to all the PGs identified was similar, but the addition of platelet lysates caused at least a sevenfold stimulation of all PGs by the corpora lutea, the greatest stimulation in production occurring in the area corresponding to PGE2 and 15-keto-PGF2 alpha. Adipose tissue by itself produced more PGs than corpus luteum (CL); but, in contrast, such production was inhibited by addition of platelet lysates to the levels of conversion of the unstimulated CL. 125I-labeled platelets bound specifically to monkey CL, but not to adipose tissue. The interaction of the platelets with the CL in the formation of PGs may be of significance in the regulation of the luteal life span.

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.

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.

Oocyte donation and gamete intrafallopian transfer in premature ovarian failure.

Gamete intrafallopian transfer (GIFT) was performed in eight patients with premature ovarian failure (POF), using donated oocytes. The steroid replacement protocol consisted of the administration of increasing dosages of 17 beta-estradiol (E2) and progesterone (P). Hormonal replacement was maintained until day 100 of gestation. All patients underwent an evaluation cycle in which serum levels of E2 and P were monitored and an endometrial biopsy was performed on day 21 or 22. All cases of GIFT were performed between days 12 and 15. Six clinical pregnancies were achieved in eight cycles (75% success rate). Three patients delivered and three are in their second or third trimester. No ectopics or miscarriages occurred. These results offer a promising approach for the establishment of fertility in agonadal patients.

Tubal embryo transfer as a treatment for infertility due to male factor.

Transvaginal follicular aspiration (TVA) with ultrasonically guided needles allows the transfer of in vitro generated embryos to the fallopian tubes (TET), performing only one surgical procedure in the process. Up to now, this approach has been used to treat 16 couples with infertility due to severe male factor. Follicular development was induced with a combination of clomiphene citrate and human menopausal gonadotropin (hMG) or follicle-stimulating hormone and hMG. Follicles were aspirated by TVA 36 hours after an injection of human chorionic gonadotropin 10,000 IU intramuscularly. A total of 169 oocytes were recovered (10.5 +/- 6.9 X +/- SD) from the 16 patients. There was failure of fertilization in 6 cases. In the remaining 10, a TET was performed 44 to 50 hours after TVA, utilizing embryos at the pronuclear stage. Six pregnancies resulted from the 10 transfers. This technique combines the advantages of proof of fertilization with a more adequate tubal embryo development and entrance to the uterine cavity that may determine and increase chance of implantation.

Frozen zygote intrafallopian transfer: a successful approach for transfer of cryopreserved embryos.

OBJECTIVE: To assess whether frozen ET to the fallopian tube is a possible alternative for cryopreserved embryos. DESIGN: Fifty-four patients (mean age 35 years) participated, in which their embryos were cryopreserved in 1.5 M propanediol at the pronuclear or two-cell stage. Each patient then underwent a steroid replacement cycle consisting of oral micronized 17 beta-E2 2 mg on days 2 to 4, 4 mg on days 5 to 7, 6 mg on days 8 to 10, and 8 mg from day 11 on. Serial ultrasounds were performed to evaluate the endometrium until an optimal thickness of > or = 10 mm triple layer was achieved. At this time, 100 mg IM progesterone was initiated and the zygote intrafallopian transfer (ZIFT) procedure was performed on the third day of P administration. The average number of embryos transferred was 4.4. RESULTS: Twenty-two clinical pregnancies resulted, giving a pregnancy rate of 41%. Eight miscarriages occurred and one ectopic pregnancy resulted, giving a live birth rate of 24%. Implantation rate was 10.8%. The highest chance of pregnancy was seen in patients who never had a previous IVF, GIFT, or ZIFT (61%). CONCLUSION: This is the first report of a series of frozen ETs to the fallopian tubes. These results indicate that tubal transfer may offer a protective benefit of the tubal environment and avoidance of endometrial trauma, and should be added to our armamentarium of replacing cryopreserved embryos.

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.

Preliminary experiences with gamete intrafallopian transfer (GIFT).

This article describes the first series of patients to undergo gamete intrafallopian transfer (GIFT) as a treatment for infertility. Ten patients with the diagnosis of either unexplained infertility or male factor were treated with human menopausal gonadotropin and human chorionic gonadotropin before surgery by laparoscopy or minilaparotomy. Semen was collected 2.5 hours before oocyte pickup at surgery and treated by the technique of wash and swim-up. After gamete evaluation, one or two oocytes and 100,000 actively motile sperm were loaded into a catheter and introduced through the fimbria. The contents of the catheter were gently emptied at a site approximately 1.5 cm inside each fallopian tube. Patients received progesterone in oil, 12.5 mg/day, from day 4 after GIFT until up to 8 weeks of gestation. Four patients became pregnant: two pregnancies aborted; the other two pregnancies proceeded to the delivery of viable infants. GIFT may be considered as an alternative to in vitro fertilization in infertility cases in which at least one fallopian tube is patent.

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 Tyrode's 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.

Embryo implantation rates in oocyte donation: a prospective comparison of tubal versus uterine transfers.

OBJECTIVE: To compare pregnancy and implantation rates in tubal and uterine transfers during a hormonal replacement cycle in an oocyte donation program. DESIGN: Prospective randomized. PATIENTS: Forty-two consecutive patients who entered an oocyte donation program. INTERVENTIONS: Twenty-two patients were assigned for uterine transfer and 20 for tubal embryo transfer (ET). RESULTS: Twenty-three pregnancies were achieved, 12 (54.5%) after uterine transfers and 11 (57.9%) after tubal transfers. Implantation rates in both groups are not significantly different (17.4% uterine transfers versus 21.5% tubal ETs). CONCLUSIONS: Our results suggest that in hormonal replacement cycles (uniform endometrial stimulation) there is no advantage in transferring embryos to the fallopian tube. Furthermore, embryo quality and endometrial receptivity appear to be significantly more important than the time of entrance of an embryo to the uterine cavity in determining its chances of implantation.

The role of the laboratory in the handling of epididymal sperm for assisted reproductive technologies.

This report describes the historical experience of our laboratory in the handling and processing of epididymal sperm from men with congenital absence of the vas deferens. Three different methods of sperm preparation were used: swim-up or resuspension in the first group of patients treated, mini-Percoll in the second group, and mini-Percoll used in conjunction with pentoxifylline and 2' deoxyadenosine in the third group. Twenty-four percent of cases achieved fertilization in group I, whereas in groups II and III the rate was 59% and 57%, respectively. Fertilization rates in each group were 6.5% in group I, 16% in group II, and 35% in group III. Three pregnancies were obtained in group I, 13 in group II, and 2 in group III. Groups II (21%) and III (63%) were able to have excess embryos to cryopreserve.