Urinary oestrogen levels and follicle ultrasound measurements in clomiphene induced cycles with an endogenous luteinizing hormone surge

Total oestrogen in urine and the ultrasonic size of the follicles were measured in relation to the onset of the endogenous luteinizing hormone (LH) surge (day 0) in 18 cycles induced with clomiphene citrate in an in-vitro fertilization programme. Oestrogen values in urine (microgram/24 h) increased progressively during the late follicular phase up to the day of the onset of the surge. The mean maximum follicle diameter (22.3, SD 4.7 mm) estimated by ultrasound was measured on day 0. At the onset of the LH surge, the values of urinary total oestrogen showed a better relation with the total volume of the first three follicles in order estimated by ultrasound (r = 0.71) than with the mean ultrasonic diameter of the leading follicle (r = 0.56). A wide range of individual values for both urinary oestrogen and follicle size was found. In another group of 32 women treated with clomiphene for recovery of oocytes used for research purposes, a good correlation was found between the mean ultrasonic follicle diameter 16 h before the laparoscopy and the follicle diameter calculated from the fluid volume at aspiration (r = 0.80). These results suggest that the decision when to give human chorionic gonadotrophin (hCG) in an in-vitro fertilization programme remains arbitrary in many individual cases. Apart from the oestrogen levels, the calculation of the size of all follicles, instead of only the leading one, may give further help in timing the hCG.     

Reduced in-vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase

Summary. A series of 62 women were managed in the University of Western Australia/PIVET Laboratory in-vitro fertilization programme. In 60 of them follicle growth was stimulated with clomiphene citrate' with or without additional human menopausal gonadotrophin (hMG) and in two with hMG alone. Follicles were aspirated at laparoscopy following an hCG trigger injection and occasionally following a spontaneous luteinizing hormone (LH) surge. Oocytes were inseminated with 0·5×10⁵−10⁵ sperm/ml 3–6 h later. A significant reduction ( P <0·001) in the fertilization rate of mature oocytes was observed in those patients whose basal serum LH values were >1 SD above the mean. Fifty-nine women subsequently had embryo transfer and of 10 clinical pregnancies, none occurred in those with elevated LH values. Reduced fertilization may be a reflection of premature oocyte maturation or ageing. This may have clinical implications in the management of some patients with unexplained infertility.     

Luteal phase after ovarian hyperstimulation

The luteal phase was investigated in 17 women with normal menstrual cycles and tubal infertility who were superovulated with clomiphene (9 cycles), clomiphene plus pulsatile human menopausal gonadotrophin (hMG) (12 cycles) and clomiphene plus pulsatile follicle stimulating hormone (FSH) (11 cycles) during an in-vitro fertilization programme. Follicles were aspirated 34-36 h after the onset of the endogenous LH surge. Urinary total oestrogen levels during the first 6 days of the luteal phase were significantly higher, the duration of the luteal phase was significantly shorter and the luteal levels of urinary pregnanediol were significantly lower in the two combination treatment cycles than in the clomiphene only cycles. When the three treatment groups were combined the mid-luteal peak pregnanediol levels and the duration of the luteal phase showed significant negative correlations with plasma or urinary oestrogen levels during the follicular and the luteal phase. It is suggested that the luteal function in cycles superovulated with clomiphene/hMG or clomiphene/FSH is disrupted and this is related to the high amounts of circulating oestrogen.     

Reduced in-vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase

A series of 62 women were managed in the University of Western Australia/PIVET Laboratory in-vitro fertilization programme. In 60 of them follicle growth was stimulated with clomiphene citrate with or without additional human menopausal gonadotrophin (hMG) and in two with hMG alone. Follicles were aspirated at laparoscopy following an hCG trigger injection and occasionally following a spontaneous luteinizing hormone (LH) surge. Oocytes were inseminated with 0.5 X 10(5)-10(5) sperm/ml 3-6 h later. A significant reduction (P less than 0.001) in the fertilization rate of mature oocytes was observed in those patients whose basal serum LH values were greater than 1 SD above the mean. Fifty-nine women subsequently had embryo transfer and of 10 clinical pregnancies, none occurred in those with elevated LH values. Reduced fertilization may be a reflection of premature oocyte maturation or ageing. This may have clinical implications in the management of some patients with unexplained infertility.     

Characteristics of patients with and without gonadotropin surges during follicular recruitment in an in vitro fertilization/embryo transfer program

Fifteen ovulatory patients undergoing ovarian stimulation with clomiphene citrate-human menopausal gonadotropin-human chorionic gonadotropin (hCG) for in vitro fertilization were studied. All 15 attained peak estradiol (E2) levels of greater than 600 pg/ml. Eight patients had an endogenous luteinizing hormone (LH) surge before the administration of hCG. The characteristics of these "surge" patients were compared with those of the remaining seven "nonsurge" patients. There was no significant difference in the peak morning E2 achieved before hCG or the endogenous LH surge, nor in the peak absolute increase in E2 over a 24-hour period in these two groups. The surge group had significantly higher E2 levels per follicle greater than or equal to 15 mm, measured by ultrasound on the morning of the day of administration of hCG or the LH surge (P less than or equal to 0.005). In addition, nonsurge patients had a greater number of follicles greater than or equal to 15 mm, compared with surge patients (P less than or equal to 0.05). It is hypothesized that greater quantities of nonsteroidal hormones, such as inhibin, produced by a greater number of preovulatory follicles in nonsurge patients, may block the pituitary response to hypothalamic gonadotropin-releasing hormone in the face of high and rising E2 levels.     

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.     

Simplification of the method of in vitro fertilization: Sonographic measurements of follicular diameter as the sole index of follicular maturity

As a simplification of the in vitro fertilization (IVF) procedure, repeated sonographic scanning of follicular diameters in stimulated cycles was performed, until the largest visible follicle reached a mean diameter of at least 28 mm, at which time human chorionic gonadotropin (hCG) was administered. Follicle puncture was performed either guided by sonography or by laparoscopy 34–36 hr after the hCG injection, and following insemination oocytes were cultured for 48–60 hr before embryo transfer (ET). Serum levels of luteinizing hormone (LH), prolactin (PRL), and estradiol-17 (E₂) at the time of hCG injection as well as follicular fluid levels of follicle stimulating hormone (FSH), LH, and PRL as well as progesterone (P), testosterone (T), and E₂ at the time of puncture were determined in retrospect using specific radioimmunoassay (RIA). It was found that the knowledge of a single value of LH or E₂ in serum should have been of little additional value for the decision to give hCG in all stimulation models used: clomiphene+hCG, human menopausal gonadotropin (hMG)+hCG, and clomiphene+hMG+hCG. The concentrations of the three steroids measured in follicular fluid were, in a high proportion of the follicles, within a normal range in all stimulation models. The group receiving clomiphene+hMG+hCG appeared promising through its higher proportion of estrogenic follicles. It is concluded that sonographic measurement of follicular diameters may be used as the sole index of follicular maturity in an IVF program. In cases with more pronounced superovulation as may be obtained by hMG stimulation, serial E₂ analyses are required in addition in order to ensure a high proportion of estrogenic follicles and an optimal pregnancy rate.     

In vitro fertilization: determination of follicular maturation for timing of human chorionic gonadotropin administration

Hyperstimulation protocols for IVF require critical timing of hCG administration to complete follicular maturation. We studied 170 IVF cycles in 142 patients and retrospectively compared ultrasound and endocrine parameters in pregnancy cycles and spontaneous LH surge cycles with nonpregnancy, non-LH surge cycles. Twenty-six of the 170 cycles resulted in a pregnancy (15.2%). Oocytes were recovered from 27 of 31 cycles in which a spontaneous LH surge occurred, and 9 of these cycles produced a pregnancy (33%). Follicle number and serum E2 on the day of ultrasound (cycle day 12 or 13) was lower in the LH surge group and pregnancy group than in the nonpregnant, non-LH surge group. In contrast, on the day of hCG administration or LH surge, serum E2 concentration per follicle greater than 1 cm in diameter was higher in the combined LH surge/pregnancy group (1219 pmol/l) than in the nonpregnancy/non-LH surge group (932 pmol/l). Our data suggest that it may be possible to individualize hCG administration at midcycle by determining the number of follicles greater than 1 cm by ultrasound on cycle day 12 or 13 and giving hCG when serum E2 levels reach 1100 to 1200 pmol/l per follicle.     

Relation between pregnancy-associated plasma protein A (PAPP-A) in human peri-ovulatory follicle fluid and the collection and fertilization of human ova in vitro

Summary. In 42 patients, blood-free peri-ovulatory follicular fluid was aspirated from 86 follicles for estimation of pregnancy-associated plasma protein A (PAPP-A) and related to several variables associated with the fertilization of human ova in vitro . Follicle growth was stimulated by clomiphene citrate alone or in combination with human menopausal gonadotrophin (hMG) and ovulation was triggered by human chorionic gonadotrophin (hCG) except in six patients who had a spontaneous surge of luteinizing hormone (LH). PAPP-A was detected in 83 of the 86 follicles aspirated, ranging from 'undetectable' to 1044 μg/l. The concentration of PAPP-A was statistically significantly correlated with increasing follicle size, follicular oestradiol-17β concentration and oocyte recovery. At PAPP-A concentrations of >200 μgl, there was no correlation noted with the ability of the ovum to be fertilized, to cleave, or between PAPP-A levels in follicles from pregnant and non-pregnant cycles. There was no significant difference in the mean PAPP-A concentration for the two stimulation regimens, but concentrations were greater in follicular fluid from patients who surged spontaneously ( P<0.001 ). It appears PAPP-A levels may provide an index of follicle maturity but not of the pregnancy potential of the ovum.     

In vitro fertilization and embryo transfer as related to endogenous luteinizing hormone rise or human chorionic gonadotropin administration

After ovarian stimulation with clomiphene citrate combined with human menopausal gonadotropin for in vitro fertilization, the appearance of a spontaneous luteinizing hormone (LH) surge before fulfillment of the minimal criteria of follicular maturity (at least one follicle greater than 19 mm and serum estradiol [E2] greater than 400 pg/ml/follicle greater than 17 mm) is associated with reduced pregnancy rates. In these cases, follicles are smaller and serum E2 values are lower at the time of the LH surge. Oocyte recovery rate is reduced, embryonic anucleate fragments are more frequently observed, and the level of luteal progesterone on day 4 after oocyte retrieval is lower. Hyperandrogenic patients are more prone to display such premature spontaneous LH surge. We concluded that in case of an untimely LH rise, laparoscopy for oocyte retrieval should be cancelled.     

Relation between pregnancy-associated plasma protein A (PAPP-A) in human peri-ovulatory follicle fluid and the collection and fertilization of human ova in vitro

In 42 patients, blood-free peri-ovulatory follicular fluid was aspirated from 86 follicles for estimation of pregnancy-associated plasma protein A (PAPP-A) and related to several variables associated with the fertilization of human ova in vitro. Follicle growth was stimulated by clomiphene citrate alone or in combination with human menopausal gonadotrophin (hMG) and ovulation was triggered by human chorionic gonadotrophin (hCG) except in six patients who had a spontaneous surge of luteinizing hormone (LH). PAPP-A was detected in 83 of the 86 follicles aspirated, ranging from 'undetectable' to 1044 micrograms/l. The concentration of PAPP-A was statistically significantly correlated with increasing follicle size, follicular oestradiol-17 beta concentration and oocyte recovery. At PAPP-A concentrations of greater than 200 micrograms/l, there was no correlation noted with the ability of the ovum to be fertilized, to cleave, or between PAPP-A levels in follicles from pregnant and non-pregnant cycles. There was no significant difference in the mean PAPP-A concentration for the two stimulation regimens, but concentrations were greater in follicular fluid from patients who surged spontaneously (P less than 0.001). It appears PAPP-A levels may provide an index of follicle maturity but not of the pregnancy potential of the ovum.     

Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge

The growth of ovarian follicles was assessed with the use of ultrasound in spontaneous cycles and in cycles stimulated with clomiphene citrate (CC) alone, CC plus pulsatile human menopausal gonadotropin, and CC plus pulsatile follicle-stimulating hormone (FSH). At the time of the onset of the luteinizing hormone surge (LH), the size of the leading follicle did not differ significantly between the spontaneous and the stimulated cycles, although it was larger in the CC/FSH cycles. During the two days before the LH surge onset, the growth rate was faster in the stimulated than the spontaneous cycles. It is suggested that despite the provocation of extremely high plasma-estradiol levels and multiple follicular development, the leading follicle in stimulated cycles ovulated at a size equal to or greater than that in spontaneous cycles. The reason for the higher follicle size in the CC/FSH cycles is, as yet, unclear.     

Ovulation triggering in clomiphene citrate-stimulated cycles: Human chorionic gonadotropin versus a gonadotropin releasing hormone agonist

Purpose To compare the use of human chorionic gonadotropin (hCG) to a gonadotropin releasing hormone (GnRH) agonist, nafarelin, in initiating ovulation and supporting the luteal phase after priming with clomiphene.Methods In 26 infertile women 50 mg clomiphene citrate produced a preovulatory-size follicle. Then, 11 women were randomized to receive two 400-g doses of nafarelin intranasally 16 h apart, and 15 women were injected intramuscularly with 5000 IU of hCG (luteal day 0 = LD0). Starting on LD6, 7 more 400-g doses of nafarelin were repeated on an every 16-h schedule or a single 2500 IU dose of hCG was given, respectively. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂), progesterone (P), and hCG were measured. On LD13, endometrium was evaluated with ultrasonography and biopsy in 19 nonpregnant women.Results As judged by a threefold rise in serum LH, an LH surge was detected on LD1 in all 11 nafarelin patients, but in only 8 hCG patients (P = 0.01). LH and FSH levels were significantly higher on LD1, 7, and 8 and were significantly suppressed on LD13 in the nafarelin group. All patients had mid-luteal P levels greater than 10 ng/ml and luteal phases longer than 13 days. Significantly different luteal E₂ or P levels were noted only on LD13, with lower values in the nafarelin group. Pregnancies were achieved in 3 of 11 nafarelin cycles and 2 of 15 hCG cycles. Luteal phase defects were also similar: 4 of 8 nafarelin patients and 7 of 11 hCG patients.Conclusion Nafarelin or hCG in conjunction with clomiphene can result in viable pregnancies, but is associated with low pregnancy rates and a high incidence of luteal phase defects.     

Precise day of ovulation determined by real-time ultrasound evidence of graafian follicular development

Real-time ultrasonic scanning was performed in 21 infertile Japanese women during 37 menstrual cycles. The maximum diameter prior to ovulation was 23.3 ± 2.9 mm in spontaneous ovulation cycles, 29.6 ± 5.2 mm in case of clomiphene therapies, and 26.7 ± 3.9 mm in HMG-HCG therapies, respectively. Size of the graafian follicles was maximum at almost the same time as the LH peak in the plasma and urine, respectively. The LH peak in the urine was determined by the hemagglutination inhibition assay, the results of which were obtainable within 2 h. Four patients became pregnant (19.0%). There was no statistical correlation between the diameter of the largest follicle and the plasma estradiols (r = 0.28, 0.2 < P < 0.3) or between the diameter of the largest follicle and the peak luteinising hormone level (r = 0.27, 0.3 < P < 0.4). Therefore, the combination of the real-time ultrasound and a hemagglutination inhibition assay for LH in urine can be clinically applied to detect the precise day of the ovulation.     

Preovulatory serum estradiol-17 beta values and ultrasound scans in 17 pregnancies which followed in vitro fertilization and embryo transfer after treatment with clomiphene and hCG

17 patients whom we treated with clomiphene and hCG became pregnant after in vitro fertilization (IVF) and embryo transfer (ET). We now report on our experience with preovulatory estradiol 17 beta values and ultrasonography in determining the time of hCG administration in these patients. The mean estradiol value just before the hCG injection was 329 +/- 75 pg/ml/follicle. The mean follicle diameter on ultrasound was 23.4 +/- 1.5 mm on the same day. In four cases, we only used estradiol values and in one case only ultrasound for the timing of the hCG injection. Both methods of monitoring follicular growth are discussed.     

Luteinizing hormone bioactivity and variable responses to clomiphene citrate in chronic anovulation

In 18 women with infertility and chronic anovulation with normal gonadotropins, three different responses were observed to increasing doses (250 to 750 mg) of clomiphene citrate (CC). Follicle development and ovulation in 8, follicle development but no ovulation without human chorionic gonadotropin (hCG) in 6, and no response to CC in 4. Serum concentrations of bioactive luteinizing hormone (bioactive-LH), immunoactive (immunoactive-LH), follicle-stimulating hormone, and estradiol (E2) were measured and follicle growth was assessed by daily ultrasound. Findings were compared with 8 normal ovulatory controls. Folliculogenesis on CC therapy, based on our data, was 78%; however, only 44% ovulated spontaneously, 34% required hCG for follicle rupture. There were no apparent hormonal indicators to predict responders from nonresponders. The absence of an LH surge in the presence of follicles and sustained high E2 concentrations in 34% of patients may be associated with a decreased E2 sensitivity at the hypothalamic-pituitary level. Ultrasound easily identified patients who responded to CC with folliculogenesis but did not initiate an LH surge. Follicle rupture was achieved promptly by hCG administration.     

Transvaginal ultrasonic assessment of endometrial growth in spontaneous and hyperstimulated menstrual cycles

Summary. Endometrial thickness and reflectivity were assessed by transvaginal ultrasound in both spontaneous and hyperstimulated menstrual cycles. Two groups of women with ovulatory cycles were examined; women in group 1 had unexplained infertility and women in group 2 were having artificial insemination by donor because of reduced spermatogenesis; a third group (group 3) comprised women with tubal infertility undergoing hyperstimulation for in-vitro fertilization. There was no difference in endometrial thickness or reflectivity between the three groups. A basic pattern of endometrial appearance common to all cycles was found, consisting of hypoechoic, isocchoic and hyperechoic images, occurring in the early follicular, late follicular and luteal phases, respectively. In all three groups a positive correlation was found between proliferative phase plasma oestradiol concentration and endometrial thickness. Group 1 r = 0·403, P<0·01; group 2 r = 0·439, P<0·01; and group 3 r = 0·617, P<0·01. There was a progressive increase in endometrial growth throughout the normal cycle until a plateau was reached 5 days after the LH surge. This pattern was also seen without acceleration of the process in hyperstimulated cycles, despite supranormal levels of oestrogen. Assessment of endometrial thickness is not a useful variable in monitoring hyperstimulated cycles. No aberrations of endometrial growth or pattern were observed in the women with unexplained infertility.     

Failure of human oocyte release at ovulation

Among 150 patients admitted for ovum aspiration, in vitro fertilization, and embryo transfer in Perth, Western Australia, 14 were found to have had at least one ovulated follicle at the time of laparoscopy. Based upon ultrasound estimation of follicle diameter 24 hours previously, ovulation occurred in 6 of 22 follicles less than 1.7 cm in mean diameter and in 16 of 19 follicles greater than 1.7 cm in mean diameter. When the ruptured follicles were flushed with heparinized fertilization medium, oocytes were collected in 13 of the 22 dispersed follicles. Twelve oocytes developed pronuclei 16 hours after the addition of spermatozoa, and 11 cleaved to the 4-cell stage within 44 hours of insemination. Oocytes were recovered from 6 of 11 patients stimulated with clomiphene alone and from all 3 patients stimulated with clomiphene supplemented with human menopausal gonadotropin. These observations suggest that oocyte release and follicle rupture are not necessarily synonymous events and that the incidence of retained oocytes in ovulated follicles following stimulation with clomiphene or clomiphene plus human menopausal gonadotropin could be on the order of 60%.     

Hormonal and ultrasonic characteristics of 23 clomiphene citrate stimulated cycles resulting in pregnancies after in vitro fertilization and embryo transfer

In vitro fertilization (IVF) and embryo transfer (ET) were successful in 24 clomiphene citrate stimulated cycles in 22 patients. Ovarian follicular growth was monitored by ultrasound (US) and serum estradiol (E2) concentrations in order to determine the most favorable time for the induction of ovulation by administration of human chorionic gonadotropin (hCG). The day of the hCG injection was considered day 0. After an almost linear growth the mean diameter of the dominant follicle reached 19.3 +/- 2 mm on day 0. Serum E2 concentrations also increased in almost linear fashion up to a maximum of 1,037 +/- 322 pg/ml on day +1. On day 0 the mean serum E2 concentration was 862 +/- 279 pg/ml. A mean serum E2 concentration per dominant and codominant follicle of 328 +/- 100 pg/ml was found on day 0. There was no linear correlation between the follicular diameters and the serum E2-values on the same day. The time of ovum recovery was often different to the time at which ovulation would have been expected in a normal cycle.     

Combined clomiphene citrate (CC) and human menopausal gonadotropins (hMG) in a fixed schedule for follicular recruitment during in vitro fertilization: Effects on premature luteinizing hormone (LH) surges

Summary Recognizing the association between plasma progesterone (P) and an imminent LH surge, we investigated the frequency of P elevation in our stimulation protocol (clomiphene citrate-human menopausal gonadotropins). P was measured retrospectively on the day before, the day of, and the day after human chorionic gonadotropin hCG administration. The means ±2 standard deviations for P were as follows: day before hCG, 0.58±0.86; day of hCG, 0.64 ± 0.56; and day after hCG, 1.47 ± 1.22. Seven patients (8%) had P levels greater than 2 SD above the mean.Significant P elevations prior to hCG are infrequent with this protocol, and in the few patients with raised P a trend toward lowered fertilization was noted. The low incidence of P elevation may be secondary to the administration of hCG in a timely manner on the sixth day of estradiol (E₂) rise.