Direct inhibitory ovarian effects of prolactin in the process of ovulation

The present study was designed to determine the effects of prolactin (PRL) on the process of follicle rupture and oocyte maturation using an in vitro perfused rabbit ovary model. In the first experiment ovaries were perfused for 12 hours with or without PRL at 10(2) or 10(3) ng/ml. Ovulation did not occur in any ovaries in the absence of gonadotropin. The majority of follicular oocytes did not progress beyond the germinal vesicle stage following treatment with PRL. The percentage of follicular oocytes which showed evidence of degeneration was also comparable in both groups. The concentrations of progesterone in the perfusate did not differ significantly between PRL-treated and control ovaries. In the second experiment, ovaries were perfused with or without PRL at 10, 10(2), or 10(3) ng/ml. Thirty minutes later 50 IU of human chorionic gonadotropin (hCG) was added to the perfusate of all ovaries. The addition of PRL to the perfusate inhibited hCG-induced ovulation in a dose-related fashion. The degree of ovum maturity and degeneration was comparable in the two groups. PRL did not affect hCG-stimulated progesterone production by the perfused rabbit ovaries. The present study demonstrates that PRL acts directly on the ovary to influence the process of ovulation, resulting in the inhibition of hCG-induced follicle rupture. These data suggest that PRL inhibits ovulation by mechanism(s) independent of ovarian progesterone synthesis.     

Progesterone protects oocytes from premature degeneration within the follicle

The present study was designed to determine the effects of gestrinone (R2323) in the process of follicle rupture and oocyte maturation and degeneration in an in vitro perfused rabbit ovary model. In the first experiment, R2323 at 10(2), 10(3), or 10(4) ng/ml was added to the perfusate of one ovary. The contralateral control ovary was perfused simultaneously with medium alone. Thirty minutes after the onset of perfusion, 50IU of human chorionic gonadotropin (hCG) was added to the perfusate of both ovaries. All ovaries exposed to R2323 plus hCG or hCG alone ovulated. The addition of R2323 to the perfusate did not affect the ovulatory efficiency of ovaries treated with hCG. No significant difference in the percentage of ovulated ova or follicular oocytes demonstrating germinal vesicle breakdown was seen with R2323 treatment. R2323 increased the degeneration rate of ovulated ova in a dose-dependent fashion. In the second experiment, in which experimental ovaries were perfused with R2323 (10(4) ng/ml) plus progesterone (10(3) ng/ml) and the control ovaries with R2323 (10(4) ng/ml) alone ovulation occurred in response to hCG. However, the addition of progesterone to the perfusate reduced the degeneration-inducing effect of R2323 on both ovulated ova and follicular oocytes. In conclusion, R2323 appears to act as an antiprogesterone, thereby promoting the degeneration of oocytes. The increased production of progesterone in the preovulatory follicle following the gonadotropin surge protects oocytes from premature degeneration within the follicles.     

Gonadotropin-releasing hormone agonists induce meiotic maturation and degeneration of oocytes in the in vitro perfused rabbit ovary

The present study was undertaken to assess the effects of gonadotropin-releasing hormone agonists (GnRH-a, buserelin and leuprolide acetate [LA]) on ovulation, oocyte maturation and degeneration, and steroid and prostaglandin production in the perfused rabbit ovary preparation. Ovulation did not occur in any of ovaries treated with buserelin or LA (10(2) to 10(4) ng/mL) in the absence of gonadotropin. Gonadotropin-releasing hormone agonists were associated with the resumption of meiosis in follicular oocytes in a dose-related manner. Furthermore, the addition of GnRH-a to the perfusate significantly increased the percentage of follicular oocytes that showed evidence of degeneration compared with contralateral untreated or human chorionic gonadotropin-treated controls. Prostaglandin E2 and prostaglandin F2 alpha production by the perfused rabbit ovaries were stimulated significantly by GnRH-a treatment. Exposure to GnRH-a failed to increase either progesterone or estradiol production by the perfused rabbit ovaries. These data demonstrate that GnRH-a act directly in the rabbit ovary to trigger meiotic maturation in oocytes within the follicles, concomitantly increasing oocyte degeneration.     

Effect of the exposure of intrafollicular oocytes to clomiphene citrate on pregnancy outcome in the rabbit

This study was designed to determine if exposure of rabbit intrafollicular oocytes to clomiphene citrate (CC) affects pregnancy outcome after in vitro ovulation, in vitro fertilization (IVF), and embryo transfer (ET). Ovaries were perfused in the presence or absence of CC (10(-5) M) and estradiol (E2, 100 ng/ml). Human chorionic gonadotropin (hCG, 50 IU) was added to the perfusate of all ovaries. In vitro ovulated ova were retrieved, inseminated, and transferred to host rabbits. Neither CC nor CC + E2 significantly affected hCG-induced ovulation or fertilization. CC significantly reduced (P less than 0.025) percentage of ovulated ova resulting in offspring. Addition of E2 significantly (P less than 0.05) reversed the reduction in offspring associated with CC alone. These results may be due to the antiestrogenic effects of CC on the intrafollicular oocyte, which compromises postfertilization development.     

Estradiol reverses the limiting effects of clomiphene citrate on early embryonic development in the in vitro perfused rabbit ovary

This study examined whether the addition of estradiol (E2) to the perfused rabbit ovary would reverse the deleterious effects of clomiphene citrate (CC) on early embryonic development. Ovaries were perfused with CC (10(-5) M) or CC + E2 (1 to 1000 ng/ml). Human chorionic gonadotropin (hCG, 50 IU) was added to the perfusate of each ovary. In vitro ovulated ova in cumulus were retrieved and inseminated in vitro. E2 significantly increased the percentage of ovulated ova achieving (1) the 2-cell stage at 36 hours, (2) the morula stage by 84 hours, and (3) the blastocyst stage at 132 hours. The percentage of inseminated ova showing evidence of degeneration was reduced in ovaries treated with E2. These data suggest that CC may exert an antiestrogenic effect on the intrafollicular oocyte, which interferes with postfertilization development.     

The effects of proteolytic enzymes on in vitro ovulation in the rabbit

The involvement of proteolytic enzymes in follicle rupture was assessed by use of the in vitro perfused rabbit ovary. Streptokinase (10 and 100 units/ml) induced ovulation in the absence of gonadotropin. Ovulation failed to occur in contralateral control ovaries. The time of ovulation in streptokinase- and human chorionic gonadotropin--treated ovaries was similar, but significantly more ova from streptokinase-treated ovaries were immature (p less than 0.001). Other ovaries were pretreated with trans-4-(aminomethyl)-cyclohexane-carboxylic acid, an inhibitor of the conversion of plasminogen to plasmin, and then perfused with human chorionic gonadotropin (50 IU). Ovulatory efficiency was significantly reduced by trans-4-(aminomethyl)-cyclohexane-carboxylic acid at 10 or 1 mmol/L (p less than 0.001), but ovum maturity was unaffected. Aprotinin (100 or 10 micrograms/ml), a potent inhibitor of plasmin, significantly inhibited human chorionic gonadotropin-induced ovulation (p less than 0.001) but did not affect oocyte maturation. Scanning electron microscopy of detergent-treated streptokinase-perfused ovaries revealed loosening and decomposition of collagen in the tunica albuginea. These results suggest proteolytic enzyme involvement in follicle rupture.     

Effect of clomiphene citrate on in vitro ovulated ova

The effect of clomiphene citrate (CC) on developmental capacity of ovulated ova was studied with isolated in vitro perfused rabbit ovaries. Fifty-two ovulated ova were recovered from ovaries perfused with human chorionic gonadotropin (hCG) in a medium containing CC and 45 ova from ovaries perfused with hCG in a CC-free medium. Ova were cultured and inseminated with capacitated sperm and observed serially for evidence of fertilization and stage of development. CC did not affect the fertilization of ovulated ova. However, the percentage of ova which had reached the morula stage by 60 hours was significantly reduced in the CC-treated (15.4%) group, compared with the control group of ovaries (48.9%). A significant percentage of inseminated ova from CC-treated ovaries (65.4%) showed evidence of degeneration, as compared with control ovaries (37.8%). Thus, a partial loss of developmental capacity may explain the discrepancy observed between the rate of successful ovulation induction and the establishment of pregnancy associated with CC administration in humans.     

Effects of prostacyclin on ovulation and microvasculature of the in vitro perfused rabbit ovary

Prostacyclin (prostaglandin I2) methyl ester at 0.1, 1, or 10 micrograms/ml was added to the perfusate of one rabbit ovary every 2 hours for the first 10 hours of perfusion. The contralateral ovary was perfused with medium alone. Prostacyclin methyl ester at 1 and 10 micrograms/ml induced ovulation in the absence of gonadotropin, with ovulatory efficiencies of 26.7% +/- 3.8% and 46.5% +/- 5.0%, respectively. Most ovulated ova (77.4%) did not progress beyond the germinal vesicle stage, and there was no significant degeneration of ovulated ova or follicular oocytes. Examination of the follicular microvasculature 5 hours after exposure to prostacyclin revealed marked vessel dilatation and filling defects at the apex. By 7 hours after prostacyclin exposure, the intrafollicular space contained extravasated resin, reflecting increased vascular permeability. The vascular changes paralleled those previously observed in gonadotropin-induced ovulation. These results indicate that prostacyclin acting locally alters the vascular integrity of the follicle wall and facilitates follicle rupture.     

A comparative study of three ovulation induction protocols in polycystic ovarian disease patients in an in vitro fertilization/embryo transfer program

Purpose This study compares the results of three ovulation induction protocols in polycystic ovarian disease (PCOD) patients undergoing an in vitro fertilizationembryo transfer (IVF-ET) program. A total of 85 cycles was studied. The patients were treated with clomiphene citrate (CC) plus human menopausal gonadotropin (hMG) (CC/hMG group), with purified menofollitropin (pFSH) plus hMG (pFSH/hMG group), and with pFSH/hMG plus gonadotropin releasing hormone analogue (GnRH-a) (analogue group). In the analogue group the suppression of luteinizing hormone (LH) with GnRH-a decreased the number of follicles <12 mm on the day of human chorionic gonadotropin (hCG) administration and the number and percentage of immature oocytes retrieved and increased the percentage of mature oocytes retrieved.Results However, fertilization rates of oocytes, cleaved embryo rates, pregnancy rates following replacement, and pregnancy outcomes were not different.Conclusion Although the suppression of the hypothalamic-pituitary-ovarian axis with GnRH-a in PCOD patients improved follicular synchrony and oocyte maturity, none of the ovulation induction protocols was superior to the others with respect to pregnancy rates and pregnancy outcomes.     

Direct effect of gonadotropin-releasing hormone agonists on the rabbit ovarian follicle.

OBJECTIVE: To determine if gonadotropin-releasing hormone agonist (GnRH-a)-induced oocyte maturation and degeneration can be attributed to the direct actions on the follicle. DESIGN: Mature rabbit follicle culture. INTERVENTIONS: The mature follicles were cultured with human chorionic gonadotropin (hCG) (100 ng/mL), buserelin acetate (10(-9) to 10(-6) M), leuprolide acetate (10(-9) to 10(-6) M), or buserelin acetate (10(-7) M) with a GnRH antagonist (10(-8) to 10(-6) M) for 14 hours. MAIN OUTCOME MEASURES: The percentage of oocytes achieving germinal vesicle breakdown, the oocyte degeneration rate, prostaglandins (PG) production by mature follicles, and the frequency of fertilization and embryonic development. RESULTS: Gonadotropin-releasing hormone agonist induced the meiotic maturation of follicle-enclosed oocytes in a dose-dependent manner while concomitantly increasing oocyte degeneration. The simultaneous addition of GnRH antagonist inhibited significantly GnRH-a-induced oocyte maturation and PG production by the mature follicles. Furthermore, a GnRH antagonist reversed the oocyte degeneration rate that had been increased by GnRH-a. The rates of normal fertilization and early embryonic development were significantly reduced in the oocytes matured by GnRH-a as compared with those matured by hCG. CONCLUSIONS: Gonadotropin-releasing hormone agonist acts directly on mature rabbit follicles to trigger the oocytes to undergo meiotic maturation, but oocytes matured in vitro by GnRH-a are not necessarily cytoplasmically mature.     

Inhibition of ovulation in marmoset monkeys by indomethacin.

The effect of indomethacin on human menopausal gonadotropin/human chorionic gonadotropin-induced ovulation was studied in marmoset monkeys. Indomethacin was effective in preventing follicular rupture and ovum extrusion when administered simultaneously with gonadotropin. The production of progesterone and luteinization of the granulosa cells were not affected by indomethacin treatment. Histologic examination of corpora lutea from indomethacin-treated animals revealed the presence of entrapped oocytes surrounded by luteal cells. These results suggest that prostaglandin synthesis is required for ovulation in this species, but not for luteinization and progesterone production.     

In vitro studies of ovulation in the perfused rabbit ovary.

A system has been developed for the perfusion of the rabbit ovary in vitro. At laparotomy, the ovarian artery is cannulated and perfused with M 199 tissue culture medium containing insulin and heparin, then removed with its vascular pedicle intact. Perfusion at 37 degrees C is maintained by using a capillary oxygenator and Buchler roller pump. The functional integrity of the perfused ovary is confirmed by serial determinations of the perfusate pH, glucose and lactate concentrations, and by ovarian histology. This in vitro model was used to study the mechanism of ovulation. One group of isolated rabbits received human chorionic gonadotropin (50 IU, intravenously) and, 8 hours later, one ovary was removed and perfused; the contralateral ovary remained in situ, serving as an in vivo control. Serial observations for follicle development and rupture were made over the subsequent 7-hour interval. The occurrence of ovulation in vitro was documented by time-lapse photography. In each animal, comparisons made between the in vitro and in vivo ovary indicated that the rate and time of follicle maturation and ovulation were comparable. Ovulation occurred between 10 and 15 hours after administration of human chorionic gonadotropin in both preparations.     

Relationship between the steroid and prolactin concentration in follicular fluid and the maturation and fertilizaton of human oocytes

Seventyeight follicles and their follicular fluid were aspirated from 46 women undergoing in vitro fertilization (IVF) procedures after stimulation of the ovaries with a low-dose human menopausal gonadotropin/human chorionic gonadotropin stimulation regimen. The concentrations of estradiol (E₂), progesterone (P), testosterone (T), and prolactin (PRL) were measured in follicular fluid and related to the maturation of the oocyte-corona-cumulus complex (OCCC) and the fertilization of oocytes. Follicles containing mature oocytes had significantly higher follicular fluid E₂ and P levels than follicles with intermediate and immature oocytes. A constant decrease in PRL and T values with advancing follicular maturation was observed. Similar results were obtained when the fertilizing ability of the oocytes was examined. The gradual decline in follicular fluid PRL and T levels during follicular development was connected with increasing E₂ and P biosynthesis and therefore seems to be an important precondition for normal follicular and oocyte maturation.     

The use of pure follicle-stimulating hormone for ovulation induction in normal ovulatory women in an in vitro fertilization program

Many ovulation induction protocols for follicular development have been reported. The present study examines pure follicle-stimulating hormone (pFSH) and human menopausal gonadotropin for ovulation induction in an in vitro fertilization and embryo transfer program. The study compares the number of ampules, the level of estradiol on the day of human chorionic gonadotropin administration and at laparoscopy, the number of oocytes retrieved, fertilization, cleavage, and pregnancy rates. The peak levels of estradiol on the day of human chorionic gonadotropin administration and the day of laparoscopy were similar, although fewer ampules of pFSH were required to reach similar criteria for oocyte maturation prior to retrieval. The fertilization rates were similar, but the cleavage and pregnancy rates favored the use of pFSH. The use of pFSH may be more physiologic in orchestrating follicular steroidogenesis in normal ovulatory women in an in vitro fertilization and embryo transplant program that subsequently could produce healthier oocytes and an improvement in the pregnancy rate.     

The effect of clomiphene citrate on human preovulatory oocyte maturation in vivo

Sixty-four infertile women underwent diagnostic laparoscopy in the periovulatory period at time-bracketed intervals following their endogenous luteinizing hormone (LH) surge. Forty-eight of these women were studied during natural cycles and 16 had mild oligoovulation and were administered clomiphene citrate (CC) to regulate their cycles. No patient received human chorionic gonadotropin. No patient was undergoing either in vitro fertilization (IVF) or gamete intrafallopian transfer (GIFT). Follicle puncture was performed and the oocytes were observed immediately for stage of maturation. Oocytes obtained from follicles exposed to CC were found to require an increased interval of time to reach metaphase I compared to oocytes obtained from natural cycles (27.75±2.2 vs 22.5 hr; mean±SE). Furthermore, the interval of time required for metaphase I oocytes to achieve metaphase II was statistically significantly shortened for CC cycles (2.4 hr for CC vs 10 hr for natural cycles. Nevertheless, there was no difference between natural and CC cycles in the time interval between LH surge onset and ovulation. These in vivo findings suggest a direct effect of CC on human oocyte maturation and may help explain the wellestablished discrepancy between the relatively high ovulation rate and the relatively low conception rate in clomiphene-induced cycles.     

The effects of progesterone antagonist RU 486 on mouse oocyte maturation, ovulation, fertilization, and cleavage

The antiprogesterone RU 486 was utilized to evaluate the possible role of progesterone in ovum maturation, ovulation, fertilization, and embryo cleavage. After gonadotropin treatment, CD-1 mice received the following experimental agents: group 1, an oil vehicle; group 2, 1 mg progesterone; group 3, 1 mg antiprogesterone (RU 486); group 4, 1 mg RU 486 and 1 mg dexamethasone. Each group of animals was injected simultaneously for 2 days (concomitant with human chorionic gonadotropin and the day after coitus). Ova or embryos were obtained on day 1, 2, 3, or 4 after human chorionic gonadotropin by flushing uteri and tubes. No differences were apparent in number of oocytes ovulated, ovum maturation, or number of oocytes progressing to two-cell embryos. However, on day 3 a marked reduction in embryos retrieved from the oviduct and uterus was apparent in the RU 486-treated groups (group 1, 84; group 3.0; group 4, 17) (p less than 0.001). In addition, few cleavage stage embryos were recovered on day 4 in the RU 486-treated groups (group 1, 74; group 2, 70; group 3, 2; group 4, 0) (p less than 0.0001). Freshly ovulated cumulus masses were recovered from the oviducts on day 4 in groups 3 and 4 (coincident with resumption of the estrous cycle). In conclusion, periovulatory RU 486 injections had no effect on nuclear maturation, ovulation, fertilization, or first cleavage division. Progesterone may not have an obligatory role in these processes. However, RU 486 administration did result in a reduced number of embryos retrieved on days 3 and 4 because of either early expulsion or destruction of the embryos.     

One ovary or two: differences in ovulation induction, estradiol levels, and follicular development in a program for in vitro fertilization

The choice of clomiphene citrate (CC) and human menopausal gonadotropin (hMG) protocols for stimulation of ovarian follicular maturation has traditionally not been made with regard to the anatomic status of the pelvis. To evaluate whether hormone production and/or follicular development vary based on the number of ovaries and/or the method of stimulation, 117 cycles were reviewed. Forty-five women received CC, 29 with two ovaries and 16 with one ovary. Seventy-two women received hMG, 50 with two ovaries and 22 with one ovary. Among women receiving CC, those with two ovaries tended to have higher initial estradiol levels and a greater number of large (greater than or equal to 15 mm) follicles. Among women receiving hMG, those with two ovaries tended to have higher estradiol levels, but the number of large follicles (greater than or equal to 15 mm) was similar. With either stimulation protocol, women with two ovaries developed a higher total number of follicles than women with one ovary. The total number of follicles in women with one ovary was similar for hMG and CC stimulations. The number of oocytes recovered at laparoscopy was greater in women with two ovaries who received hMG in comparison with CC, but did not significantly vary between women with one or two ovaries who received CC nor between women with one or two ovaries who received hMG. The number of oocytes was also similar for the women with one ovary regardless of stimulation protocol.     

Role of the amplitude of the gonadotropin surge in the rat

Ovaries that contained pregnant mare's serum gonadotropin-induced preovulatory follicles were perifused for 21 hours with (1) control media, (2) 5%, (3) 30%, or (4) 85% of the gonadotropin surge. All of the gonadotropin surges induced oocyte maturation and enhanced progesterone (P) secretion. Ovulated oocytes were observed in one of four ovaries in the 85% surge group. These data demonstrate that only a small percentage of the gonadotropin surge is required to induce a maximal P secretion and oocyte maturation. Because neither P secretion nor oocyte maturation is enhanced with increasing gonadotropin levels, these two follicular responses must be mediated through a threshold-dependent mechanism such that once the threshold level is obtained, a maximum response is induced. Greater than 85% of the surge appears to be required to induce follicular rupture, suggesting that the threshold for the follicular rupture is greater than that for either P secretion or oocyte maturation.     

Clomiphene-HMG ovarian stimulation in human in vitro fertilization and embryo transfer

In a program for in vitro fertilization and embryo transfer, laparoscopies for oocyte aspiration were performed in 40 cycles in 36 normally menstruating women with irreparable tubal diseases (IVF patients) who received clomiphene citrate (CC) and human menopausal gonadotropin (hMG). An intramuscular injection of human chorionic gonadotropin (hCG) was given to all patients after completion of follicular maturation. Fourteen cycles in 13 spontaneously ovulating women (control patients), also stimulated with CC and hMG, were adequately monitored to identify the appearance of the spontaneous luteinizing hormone (LH) surge. The follicular maturation was followed by daily ovarian ultrasonographic examination and serum estradiol estimations. Just before the LH surge the diameter of the leading follicle was 20.2 +/- 0.7 (mean +/- S.E.) mm and the serum estradiol concentration per follicle was 384.1 +/- 16.3pg/ml in the control patients. In the IVF patients the former was 20.6 +/- 0.3mm and the latter was 305.8 +/- 13.3pg/ml prior to hCG administration. When the relationship of follicular size to the rates of oocytes recovery, maturation, fertilization and cleavage was examined, larger follicles (3ml less than or equal to follicular fluid volume) showed good results. Of the 152 oocytes that were recovered from these IVF patients, 96 (63.2%) were fertilized and 79 (52.0%) cleaved. Three pregnancies resulted from 35 embryo transfers.     

Measurement of noradrenaline, dopamine and serotonin contents in follicular fluid of human graafian follicles after superovulation treatment.

We measured the noradrenaline (NA), serotonin (5-HT) and dopamine (DA) contents of 47 normally maturated and 16 cystically degenerated follicular fluid samples obtained from patients involved in the in vitro fertilization and gamete transfer program. The patients were given human menopausal gonadotropin (HMG), as a superovulation treatment, and 7,500 IU human chorionic gonadotropin (HCG) to induce ovulation 34-36 h prior to the follicular puncture done by laparoscope. The NA content of the normally developed follicles was 11.4 + 8.4 micrograms/100 ml on average. For cystically degenerated follicles, the following data were obtained: 1.1 + 0.7 micrograms/100 ml (p less than 0.001). 5-HT and DA contents in the preovulatory follicles are 14.3 +/- 8.9 and 19.3 +/- 8.2 micrograms/100 ml, respectively; at the same time, 5-HT and DA contents in the cystically degenerated follicles were 12.2 +/- 6.2 and 12.7 +/- 6.8 micrograms/100 ml, respectively. They suggest that the higher amount of NA in the follicular fluid might play an important role in the mechanism of ovulation, the regulation of postovulatory tubal motility and the release of progesterone from granulosa cells.