Sequential use of clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin in human in vitro fertilization. II. Study of luteal phase adequacy following aspiration of the preovulatory follicles

The 88 patients included in the in vitro fertilization program during 113 cycles were submitted to superovulation by sequential use of clomiphene citrate, human menopausal gonadotropin, and human chorionic gonadotropin. No correlation was found between estradiol and progesterone levels during the luteal phase and estradiol on the days preceding administration of human chorionic gonadotropin. Nineteen biopsies of the endometrium were carried out. The importance of the increase of estradiol between the day before and the day of administration of human chorionic gonadotropin is positively correlated with the quality of the endometrium.     

Human chorionic gonadotropin reduces aromatizable androgens and aromatase activity in women stimulated by clomiphene citrate and human menopausal gonadotropin

Periovulatory steroidal dynamics in women undergoing ovulation induction with clomiphene citrate and human menopausal gonadotropin were studied in 31 women with tubal blockage. Serum estradiol levels were significantly reduced 36 hours after human chorionic gonadotropin administration (from 1792 +/- 162 to 926 +/- 132 pg/ml, p less than 0.001). Peripheral levels of testosterone and androstenedione did not change during this periovulatory time. Progesterone and 17 alpha-hydroxyprogesterone, as anticipated, significantly increased with an early rise noted within the first 8 hours of human chorionic gonadotropin administration (p less than 0.001). A significant reduction of the ratios of the steroidal pairs 17 alpha-hydroxyprogesterone-progesterone (17 alpha-hydroxylase) and androstenedione-17 alpha-hydroxyprogesterone (17,20-desmolase) was observed after human chorionic gonadotropin injection (p less than 0.001). Aromatase activity appeared to be inhibited because of a significant reduction in the estradiol/testosterone ratio 34 to 36 hours after human chorionic gonadotropin administration. Thus human chorionic gonadotropin, which triggers ovulation in women treated by clomiphene citrate-human menopausal gonadotropin, appears to partially reduce aromatizable substrate as well as inhibit aromatase activity.     

Timing human chorionic gonadotropin administration by days of estradiol rise

A critical aspect of human in vitro fertilization (IVF) treatment programs is the timing of human chorionic gonadotropin (hCG) administration to induce final oocyte maturation. Patients were treated with clomiphene citrate (Serophene, Serono Laboratories, Inc., Randolph, MA), 100 mg, and human menopausal gonadotropin (Pergonal, Serono Laboratories, Inc.) 1 ampule daily from cycle days 4 through 8. The baseline estradiol (E2) level was determined by averaging the E2 values from cycle days 3 and 4. The first day of E2 rise was defined as the day that the E2 level exceeded 150% of the baseline. In general, 5000 IU of hCG (Profasi-HP, Serono Laboratories, Inc.) was administered on the evening of the sixth day of E2 rise. Significantly fewer oocytes were recovered per patient when the hCG was administered on the seventh day of E2 rise, compared with the fifth or sixth day. The recovered oocytes in the seventh-day E2 group had a significantly lower fertilization rate, compared with the other groups. The use of E2-rise days proved to be a simple, successful technique for the timing of hCG administration in an IVF treatment program.     

Ovulation induction with gonadotropins in women with polycystic ovary disease

Seventy-two infertile women with polycystic ovary disease (PCOD) and clomiphene citrate treatment failure underwent 220 human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) treatment cycles for ovulation induction over a period of 19 months. Forty-two patients ovulated but failed to conceive on clomiphene, and the remaining 30 failed to ovulate on clomiphene. Monitoring of treatment consisted of serum 17 beta-estradiol (E2) levels and ultrasonic assessment of follicular growth. Treatment was withheld whenever the E2 levels exceeded 1,500 pg/mL and/or when more than two follicles greater than or equal to 17 mm in diameter each were encountered on ultrasonography. Twenty-nine patients conceived (40.2%), and 23 delivered viable infants. Twenty-three of the 29 pregnancies were achieved in the 42 patients who ovulated on clomiphene, while only 6 pregnancies resulted in the 32 anovulatory patients on clomiphene. Six patients (20.6%) aborted in the first trimester. Multiple pregnancies consisted of only two sets of twins (6.9%). There were only two cases of mild hyperstimulation (2.7%) and no severe hyperstimulation. Because of the low occurrence of multiple pregnancies and hyperstimulation and the reasonable success rate, all PCOD patients should be started on this protocol.     

Outcome of in vitro fertilization in women with low response to ovarian stimulation

The occurrence of low-response (LR) cycles (defined as peak estradiol levels less than 300 pg/ml) in an in vitro fertilization program using a 3 ampule/day human menopausal gonadotropin regimen were retrospectively reviewed. LR occurred in 51 of 564 patients (9%). The LR serum estradiol levels were categorized into four different patterns that were further analyzed for outcome in these initial cycles, as well as for their predictive value of response in subsequent in vitro fertilization cycles. Changing the stimulation protocol to a combination of clomiphene citrate and human menopausal gonadotropin did not improve the ovarian response in this group of LR patients, and their chance to complete a subsequent normal-response treatment cycle was 32%. Suggestions are made to predict outcome and govern management of women with previous LR cycles.     

Altered follicular development in clomiphene citrate versus human menopausal gonadotropin-stimulated cycles for in vitro fertilization

The pattern of periovulatory and luteal phase serum estradiol (E2) and progesterone (P) as well as follicular fluid (FF) E2, P, androgen, gonadotropin, and prolactin concentrations of eight women undergoing clomiphene citrate (CC)/human chorionic gonadotropin (hCG) stimulation and eight women undergoing human menopausal gonadotropin (hMG)/hCG stimulation of follicular development for the purpose of in vitro fertilization were compared. Ovulation was induced with either a 5-day course of CC (100 mg/day beginning on day 5 of the cycle) or an individualized hMG regimen, and laparoscopy was performed 36 hours after hCG administration. The length of the luteal phase was significantly longer (P less than 0.05) in the CC-treated group as compared with the hMG-treated group. The pattern of serum E2 levels differed significantly (P less than 0.01) in that E2 levels were lower in the early and midluteal phase in CC-stimulated cycles; in addition, a delayed second E2 peak was observed in the late luteal phase in these women. Serum P levels, however, were lower in the hMG-stimulated group. Analysis of FF hormone concentrations revealed significantly (P less than 0.05) higher concentrations of E2 and androsterone in the FF of hMG-treated patients. It is concluded that follicular development in CC-stimulated cycles differs markedly from that in hMG-stimulated cycles. These differences may reflect either an altered follicular maturational process or may represent a direct inhibitory effect of CC on follicular steroidogenesis.     

Alteration of human follicular fluid plasminogen activator activity by ovarian hyperstimulation

Recently, it has been shown that granulosa-cell secretion of plasminogen activator (PA) is responsive to luteinizing hormone (LH)/human chorionic gonadotropin (hCG) as well as follicle stimulating hormone (FSH) in the rat and the pig. Accordingly, we asked whether PA activity in follicular fluid from exogenously stimulated human follicles was different from that of normal cycles and whether or not these activities correlated with follicular maturation as determined by follicular fluid steroid concentration. Follicular aspirates were obtained from women who were participating in an in vitro fertilization protocol. Follicular fluid concentrations of estradiol and progesterone were determined by established radioimmunoassay. PA activity, determined using a modified indirect solid-phase radioassay, was significantly less in follicles from patients treated with human menopausal gonadotropin (hMG) plus clomiphene (P<0.05) compared to untreated patients or those receiving hMG or clomiphene alone. Correlations of PA activity and follicular fluid steroid concentrations demonstrated no significant correlation in samples from treated patients. In contrast, untreated spontaneously cycling patients had a significant (r=0.89,P<0.05), positive correlation between follicular fluid estradiol levels. There was no correlation between PA activity and follicular fluid progesterone levels in any of the groups. These results suggest that a subtle balance in granulosa-cell secretion of PA and steroids exists, which appears to be disrupted by follicular hyperstimulation during treatment of patients participating in in vitro fertilization protocols.Supported in part by Clinical Investigator Award HD-00401 and by the University of Southern California Faculty Research and Innovation Award.     

Endocrine changes in the late-follicular and postovulatory intervals as determinants of the in vitro fertilization pregnancy rate

This investigation examines the hormone pattern in in vitro fertilization (IVF) cycles from the time of human chorionic gonadotropin (hCG) administration through embryo transfer to ascertain whether the absolute levels or secretory patterns of the major reproductive hormones affect the IVF pregnancy rate. Thirty-one women who underwent IVF treatment were enrolled in the study. All patients received clomiphene citrate/human menopausal gonadotropin for ovulation induction. Significant elevations in serum estradiol (E2) levels in the pregnant group were found throughout the cycle interval studied. After hCG administration the serum hCG levels were not different between the groups. Significant elevations in serum progesterone (P) concentrations were found in the pregnant group from the day after laparoscopy through embryo transfer. Embryos obtained from the pregnant group appeared to be different in that the mean number of blastomeres per embryo transferred was significantly greater. Therefore for achievement of an IVF pregnancy the optimal hormone pattern employing combination ovulation induction in the ovulation to transfer interval is a relatively high E2 level in ovulation followed by a high P level at transfer and into the luteal phase. These elevated hormone levels do not depend on the response to exogenous hCG.     

Follicular monitoring and outcome of in vitro fertilization in gonadotropin-releasing hormone-agonist-treated cycles

Previous investigations of in vitro fertilization (IVF) cycles stimulated with gonadotropins have shown that it is possible to predict IVF outcome on the basis of the pattern of estradiol (E2) production in the terminal phase of follicular growth. This analysis looked at endocrine and ultrasound parameters in the late follicular phase of 320 patients in their first cycle of IVF. All cycles were stimulated by an association of gonadotropin-releasing hormone agonists (GnRH-a) and human menopausal gonadotropins (hMG). The pregnancy rate (PR) was not related to the E2 pattern in the 5 days before ovulation induction and was 33% and 39% even when E2 values fell during hMG administration. The PR was independent of the E2 level and the number of follicles greater than 14 mm on the day of human chorionic gonadotropin administration (day 0). The incidence of multiple pregnancy increased when E2 levels were greater than 1,000 pg/mL. It is concluded that follicular phase parameters used for cycle cancellation in hMG-stimulated IVF cycles cannot be extrapolated to GnRH-a/hMG cycles.     

Clinical, immunologic, and genetic definitions of primary and secondary recurrent spontaneous abortions

Follicular fluid was obtained from anovulatory patients (n = 12), stimulated with human menopausal gonadotropin, clomiphene, and human chorionic gonadotropin to evaluate the relative responses of inhibin, follicle regulatory protein, and steroid levels in follicles from ovaries requiring exogenous stimulation for follicular development. Follicular fluid concentrations of estradiol, progesterone, androstenedione, testosterone, dihydrotestosterone, and 3 alpha-androstenediol were determined by radioimmunoassay. Follicular fluid inhibin activity was determined by suppression of rat pituicyte follicle-stimulating hormone, and follicle regulatory protein activity was determined by suppression of porcine granulosa cell aromatase. The mean level of steroids were progesterone (7529 +/- 1601 ng/ml), estradiol (1082 +/- 158 ng/ml), androstenedione (15.2 +/- 3.17 ng/ml), 3 alpha-androstenediol (0.90 +/- 0.13 ng/ml), testosterone (2.23 +/- 33 ng/ml), and dihydrotestosterone (0.77 +/- 0.11 ng/ml). Follicle regulatory protein activity was 16.6% +/- 4.3% and mean inhibin level was 62.9 +/- 7.52 U. These results are in contrast to reports of follicular fluid steroid levels from normal ovulatory patients treated with exogenous gonadotropin. Although altered levels of hormones were present within these follicles, they clearly were not atretic, as evidenced by elevated estradiol levels and estradiol/androstenedione ratios. Alterations in the normal follicular response to pharmacologic gonadotropin stimulation in the follicles of anovulatory women suggest the presence of granulosa cell dysynchrony .     

Short-term luteinizing hormone-releasing hormone agonist treatment: prospective trial of a novel ovarian stimulation regimen for in vitro fertilization

Over a period of 4 months, 262 infertile couples participated in a prospective pseudorandom trial of a novel short-term luteinizing hormone-releasing hormone/human menopausal gonadotropin (LH-RH/hMG) treatment; the short-Buserelin-gonadotropin (Hoechst, Hounslow, United Kingdom) regimen. Patients treated with the short-Buserelin-gonadotropin regimen had a significantly higher likelihood of achieving pregnancy than patients treated with the standard clomiphene citrate (CC)/hMG regimen (respectively, 35.5% and 18% per treatment cycle). A significantly higher number of eggs were collected after short-Buserelin-gonadotropin treatment than CC/hMG, but the proportion of patients having a given number of embryos replaced was similar in the two groups. The short-Buserelin-gonadotropin-treated patients were distinguished from the CC/hMG-treated group by significantly lower levels of LH in the late follicular phase and a lower plasma level of estradiol. A detrimental relationship between elevated endogenous LH secretion and failure of implantation has been established. The nature of the short-Buserelin-gonadotropin regimen provokes high levels of endogenous gonadotropin secretion in the early follicular phase and induces a suppression of gonadotropin secretion in the late follicular phase. This may be the physiologic basis of the greater implantation rate after short-Buserelin-gonadotropin treatment than is seen with conventional CC/hMG treatment.     

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 day of initiation of human menopausal gonadotropin stimulation affects follicular growth in in vitro fertilization cycles

The attainment of synchronous follicular development in human menopausal gonadotropin/human chorionic gonadotropin-stimulated cycles for in vitro fertilization (IVF) continues to be a perplexing problem. Two regimens of follicle stimulation for IVF cycles were, therefore, compared. Twenty-nine patients commenced human menopausal gonadotropin (hMG) therapy on day I of the menstrual cycle (Group I), while 30 women received hMG from the third day of the cycle (Group II). The hMG therapy was tailored to the individual patients's response, based on ultrasonographic measurements of follicular size and serum estradiol (E₂) levels. Both groups of patients received a mean of 19.6±1.4 ampules of hMG over a mean of 6.1±0.2 days. The pattern of serum E₂ and progesterone levels in the periovulatory and luteal phase was not affected by the day of initiation of hMG therapy, although Group I patients demonstrated lower (P<0.05) E₂ levels on the 2 days prior to human chorionic gonadotropin (hCG) administration. In terms of follicle growth, Group II follicles consistently demonstrated a significantly (P<0.01,x ² test) larger proportion of medium- and large-sized follicles compared to Group I follicles on almost all of the days when ultrasonographic measurements were taken. In addition. Group II follicles demonstrated an earlier shift (day—1) to the larger follicles than Group I follicles (day 0). Significantly (P<0.001) more oocytes were recovered per uspirated follicle in Group II patients, but the fertilization rate per oocyte was greater (P<0.003) for Group I oocytes. Nevertheless, pregnancy rates did not differ between the two groups. It is suggested that a difference between the two groups of patients in the quantity or quality of gonadotropin receptor sites in the early part of the follicular phase may account for both the diminished E₂ production in the follicular phase and the persistent depressed follicular growth in Group I patients.     

In vitro fertilization and embryo transfer: An individualized approach to ovulation induction

Because of the inherent marked individual responsiveness to ovulatory stimulating agents, a highly individualized approach to ovulation induction for in vitro fertilization has been utilized. The starting time for medication was varied according to the previous mean cycle length. The dose of clomiphene citrate was adjusted to the body weight. The dose and duration of both clomiphene and human menopausal gonadotropin (hMG) were adjusted as early as treatment day 5 in accordance with the ultrasonic findings. The day of human chroionic gonadotropin (hCG) administration was varied according to a combination of ultrasonic findings and plasma estradiol levels. This approach resulted in a mean retrieval of four oocytes per laparoscopy, 75% of which were mature, a fertilization rate of 78%, and a pregnancy rate of 14% of laparoscopies.     

Influence of superovulation on endometrial and embryonic development

The authors have studied the temporal relationship between follicular rupture and endometrial development in 13 women during a natural ovarian cycle (length 25 to 35 days), and subsequently after standard treatment with clomiphene citrate, human menopausal gonadotropin and human chorionic gonadotropin (hCG) to induce multiple folliculogenesis for oocyte recovery, in vitro fertilization, and embryo freezing (cycle length 23 to 27 days). An endometrial biopsy was taken during both cycles 1.5 to 2.0 days after the oocytes had been released or removed. The samples were examined by light and transmission electron microscopy. Samples of peripheral blood were taken at defined times for hormone analysis. After treatment 11 subjects (85%) had advanced morphological development of the endometrium (8 women by 3 to 4 days, 3 women by 1 to 2 days). The concentrations of plasma estradiol (E2) and progesterone (P) on the days of follicular rupture and endometrial biopsy were significantly raised in the treatment cycles. The concentration of total urinary estrogens on the day of hCG administration and the mean change in the concentration of plasma E2 (treatment/control) on the days of endometrial biopsy were positively correlated with the extent of endometrial advancement. In addition, the mean change in the concentration of plasma P (treatment/control) was markedly increased on the days of follicular rupture and endometrial biopsy in those subjects with an advanced endometrium. Embryonic development was not so obviously related to the extent of superovulation. Asynchronous endometrial and embryonic development may therefore contribute to the low pregnancy rate in these patients.     

Cumulative ovulation rate in human menopausal/human chorionic gonadotropin-treated monkeys: "step-up" versus "step-down" dose regimens

Cumulative ovulation rates in monkeys given human menopausal gonadotropin/human chorionic gonadotropin (hMG/hCG) on menstrual cycle days 3 to 9 in a "step-up" versus "step-down" protocol were compared with those achieved by hMG only. Using direct ovarian observation to detect sites of follicular rupture, retrograde tubal irrigation to collect eggs or embryos, and serum estradiol and progesterone levels to infer changes in ovarian status, we counted the number of ovulations at intervals of 24, 48, 72, 96, and 120 hours after hCG or comparable intervals without hCG. The data indicate (1) whereas most ovulations occurred on or before day 3 after hCG treatment, when the "step-up" dose regimen was employed additional follicles had ruptured on days 4 and 5; (2) a "step-down" protocol for hMG therapy may better synchronize follicular rupture and reduce susceptibility to delayed ovulations compared with a "step-up" dose regimen; and (3) under hMG stimulation, few follicles would rupture spontaneously (without hCG or luteinizing hormone treatment).     

An increased initial follicle-stimulating hormone/luteinizing hormone ratio does not affect ovarian responses and the outcomes of in vitro fertilization

The tenet that a combination of human follicle-stimulating hormone (hFSH)/human menopausal gonadotropin (hMG) improves follicular recruitment was assessed by randomly treating ovulatory women either with hFSH/hMG on days 3 and 4 of the cycle followed by two ampules of hMG daily or with a constant daily dose of 2 ampules of hMG. Estradiol (E2) levels on the day of human chorionic gonadotropin (hCG) and the mean number of mature, immature and atretic oocytes per cycle did not differ between the two groups. Likewise, fertilization, cleavage, and pregnancy rates were similar for the two treatments. When daily hormone levels were compared in 11 patients during two successive treatment cycles with both stimulation protocols, the temporal pattern of FSH accumulation was repeated in both cycles, but FSH levels were significantly higher when patients received hFSH/hMG. Nevertheless, during both cycles, E2 reached similar peak levels and the mean number of follicles per cycle on the day of hCG administration was not different. We conclude that routine use of hFSH/hMG does not improve the success of an in vitro fertilization (IVF) program and that higher FSH levels do not change the individuality of ovarian response in the same woman.     

Biochemical studies following ovulation induction with clomiphene citrate and human menopausal gonadotropin in infertile Indian women

Biochemical studies were done in 65 anovulatory women before and following induction of ovulation with ethinyl estradiol/clomiphene citrate/human chorionic gonadotropin and also clomiphene citrate/human menopausal gonadotropin/human chorionic gonadotropin to determine the metabolic adverse effects, if any, of such therapy. Significant increases occurred in lipids and lipoprotein levels, while minor changes were seen in liver function enzymes and fasting blood sugar. Sialic acid decreased significantly after therapy in both the regimens, and both induction therapies seem to be free of adverse metabolic effects of hormones. On comparison with normal ovulatory women, this study suggests that some of the biochemical parameters can serve as reliable indicators of ensuing ovulation.     

Implantation,abortion, and birth after in vitro fertilization using the natural menstrual cycle or follicular stimulation with clomiphene citrate and human menopausal gonadotropin

The incidence of pregnancy and abortion was analyzed in 1679 patients having embryos replaced after oocyte recovery and in vitro fertilization in order to alleviate their infertility. In these patients, 364 pregnancies were achieved and 108 abortions occurred. Patients were treated either on their natural cycle, having a spontaneous luteinizing hormone (LH) surge to induce ovalation, or after ovarian stimulation using clomiphene citrate alone or in combination with human menopausal gonadotropin (hMG). The data were assessed in relation to the numbers of embryos replaced, the follicular stimulation and ovulation induction regime used; the dose of gonadotropins and the dose of antiestrogens, and the age of the patient. The most successful treatment for the number of live births per laparoscopy was the use of clomiphene citrate in combination with human menopausal gonadotropin followed by human chorionic gonadotropin (hCG) to induce follicular maturation and ovulation. This treatment produced a significantly higher (P<0.001) number of patients with multiple embryos (86.5%). There was no significant effect on pregnancy or abortion with alow (<400-mg) or high (400-mg) dose of clomiphene. The total dose of gonadotropins used did not influence the incidence of pregnancy or abortion. The use of hCG with hMG induced a significant (P<0.01) positive effect on the incidence of pregnancy. The incidence of pregnancy showed a progressive decline with increasing age but there was a highly significant (P<0.01) increase in the incidence of abortion with increasing age. With increasing numbers of embryos replaced, up to three, the incidence of deliveries increased. The most successful follicular stimulation regime and the replacement of three embryos produced about 25% of the patients delivering normal healthy babies after embryo replacement.     

Successful treatment of long-term unexplained infertility with gonadotropin-releasing hormone agonist analogue and human menopausal gonadotropin

Thirty-five couples with long-term unexplained infertility were treated in 98 cycles by giving gonadotropin-releasing hormone analogue, human menopausal gonadotropin and human chorionic gonadotropin for induction of superovulation. Pregnancy occurred in 24 cycles (24.5% per cycle and 68.6% per patient). All patients had been treated previously with clomiphene citrate and human menopausal gonadotropin in 110 cycles in conjunction with artificial insemination, but failed to achieve pregnancy. Pituitary suppression with gonadotropin-releasing hormone analogue followed by ovarian stimulation with human menopausal gonadotropin may correct the subtle abnormalities at the hypothalamic hypophyseal level that could be present in cases of unexplained infertility and it should be tried first for these patients before resorting to in vitro fertilization or gamete intrafallopian transfer.