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Reproductive Endocrinology |
Department of Obstetrics and Gynaecology (A.M.F., P.V., V.P., M.G., R.A., A.C., S.M.) and Department of Pediatrics (A.R.), Catholic University, 00168 Rome, Italy; and Institute for Research (A.L.), 96018 Troina, Italy
Address all correspondence and requests for reprints to: A. M. Fulghesu, Clinica Ostetrica Ginecologica, Università Cattolica del Sacro Cuore, Lgo Gemelli 8, 00168 Roma, Italy.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study included 34 consecutive women, 22–37 yr old, affected by PCOS. All patients were submitted to 52 cycles of gonadotropin treatment to achieve induction of ovulation. PCOS was diagnosed on the basis of all these criteria: clinical findings (presence of amenorrhea or oligomenorrhea and hirsutism), elevated plasma androgen levels (normal values: androstenedione (A) 2.0–7.0 nmol/L, testosterone (T) 0.6–2.0 nmol/L), and bilaterally normal or enlarged ovaries with 7–10 microcysts (<5 mm in diameter) as visualized at ultrasonography. A normal LH/FSH ratio was not considered an exclusion criterion. No patient had hyperprolactinemia (normal range of PRL 3.5–26.5 µg/L, conversion factor 1.0) or clinical evidence of hypercorticism or thyroid dysfunction. All had spontaneous onset of puberty and sexual development but suffered from chronic oligomenorrhea that was associated to sterility for a minimum period of 3 yr. All women had been treated previously with clomiphene citrate (100–150 mg/die for 5 days) for at least 6 months but none conceived. The patency of at least one tube and the presence of a normal uterine cavity was documented by hysterosalpingography and/or laparoscopy performed on all patients within 1 yr before enrollment. Partner semen analysis excluded oligozoospermia, asthenozoospermia, or teratozoospermia (<20 x 106 sperm/mL, <50% of total mobility, <30% of normal morphology of sperm cells, according to World Health Organization criteria) (8). Medical therapy, using drugs that could affect carbohydrate metabolism or plasma steroid levels, was interrupted in all patients at last 3 months before the study. The Institutional Board of the Department of Obstetrics and Gynecology approved the plane of the work; informed consent was obtained from each patient.
Clinical protocol
Patients were hospitalized during the early follicular phase (days 1–7 of the cycle) after progestin-induced menses. After 3 days of a standard 300-g carbohydrate diet, they were subjected to a 75-g oral glucose tolerance test (OGTT) and to hormonal baseline level determinations. All patients then were discharged, and, at day 3–4 of the subsequent spontaneous or progestin-induced menstrual cycle, started treatment for ovulation induction. In all patients, follicular maturation was induced using FSH (Metrodin Serono, Rome, Italy 150 IU IM/die). In case of insufficient ovarian response [estradiol (E2) < 367 pmol/L after 7 days of therapy, conversion factor 3.671], the dosage was increased by 75 IU every five days; the maximum daily dosage was 225 IU/day. When, at the least, one follicle reached a diameter of 18 mm, FSH treatment was interrupted, and 24 h later, 5000 IU of hCG (Profasi Serono Rome) were administered im (day of hCG administration was considered day 0). The ovarian response was evaluated daily at vaginal ultrasound and by determining plasma E2 levels; plasma levels of A were assayed retrospectively. Luteal progesterone assay and ultrasound examination also were performed 7 days after induction of follicular rupture. When subjects were subjected to several cycles of ovulation induction, an interval of at least 4 weeks between individual cycles was observed.
Data analysis
Body mass index (BMI) was calculated according as: body weight (kg)/height (m2). In case of a BMI greater than 25, the patient was considered to be obese. A normal glycemic response to the OGTT was defined according to the criteria of the National Diabetes Data Group (9) (normal glucose tolerance: plasma glucose at 0 min less than 115, peak value less than 200, at 120 min less than 140 mg/dl; impaired glucose tolerance: 0 min 115–140, peak value >200, at 120 min >200 mg/dL; diabetes mellitus: 0 min >140, peak value >200, at 120 min >200 mg/dL, conversion factor to SI units, 0.05551). The insulinemic and glycemic responses to the glucose load were expressed as area under the curve (AUC) calculated according to the trapezoidal rule. The insulinemic response to the OGTT was considered normal when the insulin levels were less than 717 pmol/L (conversion factor 7.175) at 30 and 60 min and when the AUC was lower than 107,550 pmol/L x 240 min, as established by the standard procedure of our laboratory. This cut-off value was obtained from a control population of 100 lean subjects studied during the early follicular phase (10). The free androgen index (FAI) was calculated as: T x 100/SHBG.
Ovarian volume was calculated using the formula for a prolate ellipsoid (0.5237 x D1 x D2 x D3, where D1, D2, and D3 are the maximal longitudinal, anteroposterior, and transverse diameters, respectively). Ovulation was ascertained when, at midluteal phase, serum progesterone values greater than or equal to 25 nmol/L (conversion factor: 3.180). The presence of ovarian hyperstimulation was evaluated by ultrasound and plasma E2 levels. OHSS was defined according to the criteria reported by Golan et al. (11). The classification proposed by these authors stresses the importance of ultrasound and the clinical features of the syndrome: mild OHSS grade 1: ovarian enlargement ranging from 5–12 cm and abdominal distension and discomfort; grade 2: features of grade 1 plus nausea, vomiting, and/or diarrhea; moderate OHSS grade 3: features of mild OHSS plus ultrasonic evidence of ascites; severe OHSS grade 4: features of moderate OHSS plus clinical evidence of ascites and/or hydrothorax or breathing difficulties; grade 5: all of the above plus change in blood volume, increased blood viscosity caused by hemoconcentration, coagulation abnormalities, and diminished renal perfusion and function.
Assays
Plasma baseline levels of gonadotropins, sex hormone-binding globulin, A, and T were determined. Insulin and glucose serum concentrations were analyzed in all samples after oral glucose challenge. During ovulation, induction E2 and A plasma levels were evaluated in all samples. Glucose levels were determined using the glucose oxydase technique. Plasma samples for hormone determinations were maintained at -20 C until assayed. All hormones were measured by RIA methods using commercial kits (Radim, Pomezia, Italy). The immunoradiometric assay (IRMA) on solid phase (coated tube), based on monoclonal double-antibody technique, was used for LH, FSH, and SHBG detection. Steroids were assayed by an RIA direct method in human serum or plasma. Insulin was assayed using an RIA method. Intraassay and interassay coefficients of variation were respectively: LH 5.6% and 9.1%, FSH 6.9% and 8.4%, E2 2.3% and 3.5%, T and A 6.1% and 9.3%, insulin 5.1% and 6.2%, SHBG 6.9% and 8.5%. Data are presented as mean ± SD.
Data distribution was tested by Kolmogorov-Smirnov test. Statistical
analysis was performed using the nonparametric Mann-Whitney
U test for the comparison of means because the data analyzed
were not normally distributed. The 
square test was used for the
comparison of percentages. The linear correlation between all variables
was performed after its logarithmic transformation. The significance
level was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the insulinemic response to glucose load, 20 patients resulted as hyperinsulinemic (14 obese and 6 lean subjects) and 14 normoinsulinemic (3 obese and 11 lean subjects). The BMI values were higher in hyperinsulinemic patients compared with normoinsulinemic ones (BMI: 27.8 ± 5.3 and 23.2 ± 4.1 Kg/m2, respectively; P < 0.05). Fasting glucose levels were similar in both groups. The age and the frequency of tubal factors also were similar in the 2 groups (age: 28.1 ± 4.4 yr in hyperinsulinemic and 29.4 ± 3.8 yr in normoinsulinemic patients; the presence of monolateral tubal factor was recognized: in five hyperinsulinemic subjects for 9 cycles (29% of cycles) and in 3 normoinsulinemic subjects for 5 cycles (23% of cycles).
Table 1
shows the clinical and hormonal characteristics
of the populations studied. There were no differences in gonadotropins
or in A and T levels. Sex hormone binding globulin levels were lower
and the FAI was higher in hyperinsulinemic patients than in the
normoinsulinemic ones.
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, fasting and insulin AUC of the two groups also are
indicated.
In Table 2, the outcome of the stimulated cycles is
indicated. Twenty-one cycles of treatment were employed in the
normoinsulinemic group patients (7 women: 2 cycles), whereas
hyperinsulinemic subjects were submitted to 31 cycles of treatment (11
women: 2 cycles). The FSH dosage necessary to achieve follicular
maturation was similar in both groups. Considering the dosage
administered in relation to body weight in each subject, no differences
were observed in the dose/BMI ratio between the two groups.
Furthermore, no differences were observed between the dosage or
dose/body weight ratio in obese and lean subjects (FSH dosage: obese
1560 ± 675, lean 1297 ± 420 UI; dose/BMI: obese 52.5
± 19, lean 60 ± 18; obese 28 cycles, lean 22 cycles). The
ovulation rate was similar, whereas the global incidence of OHSS was
significantly higher in the hyperinsulinemic group. In this group, 16
mild cases (12 grade one and 2 grade two), 2 moderate (grade three),
and 2 severe cases of OHSS occurred. In the normoinsulinemic group, we
observed mild OHSS in five cycles (4 grade one and 1 grade two) and
moderate OHSS in one cycle. No case of severe OHSS occurred in this
group of patients.
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shows the changes in ovarian volume during
gonadotropin stimulation. Basal ovarian volumes were similar in both
groups of patients; however, a higher rate of ovarian enlargement
starting 5 days before hCG injection until ovulation was noted in the
hyperinsulinemic group.
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, data also are analyzed considering the BMI of each subject.
Obese normoinsulinemic patients showed a lower rate of increase of
ovarian dimensions in response to FSH compared with lean subjects. In
the hyperinsulinemic group, on the contrary, both obese and nonobese
subjects had similar follicular growth rates during gonadotropin
administration.
In Fig. 2, ultrasonographic data concerning follicular
development during stimulation are represented. Basally, there were no
differences between the two groups. As expected, gonadotropin treatment
induced growth of several follicles in both normo- and hyperinsulinemic
patients. From day -5 until ovulation, the number of small and
immature follicles was less in the normoinsulinemic group than in the
hyperinsulinemic group. There were no significant differences in the
number of preovulatory mature follicles between the two groups.
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shows the E2 and A plasma levels in
the hyperinsulinemic and normoinsulinemic groups during the stimulation
protocol. E2 plasma levels gradually increased in both
groups; starting from day -1, significantly higher levels were
observed in hyperinsulinemic subjects compared with those observed in
the normoinsulinemic group. No differences were found when we compared
all obese patients with lean subjects. Plasma levels of A also
increased during treatment; no significant difference between the two
groups was observed.
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We also investigated the E2/A ratio as an index of aromatase activity: basally, there was no difference between the two groups (H 0.042 ± 0.015 pmol/L and N 0.034 ± 0.025 pmol/L), whereas, just before ovulation, the aromatization index of hyperinsulinemic patients was significantly higher than that of normoinsulinemic ones (0.63 ± 0.58 pmol/L vs. 0.26 ± 0.30 pmol/L; P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical management of gonadotropin-induced ovulation in PCOS is difficult, in view of the risk of developing OHSS. The ovarian response may be rapid and not related to the dosage of hormones administered. In a recent paper, Filicori et al. (14) analyzed the impact of increased insulin secretion on pulsatile GnRH ovulation induction in patients with multifollicular ovaries and PCOS. Higher insulin levels, as well as a positive correlation between insulin response to glucose load and baseline ovarian volume, were found in those patients who failed to ovulate in spite of GnRH treatment. This suggested complex interferences of insulin with follicular maturation.
The present study indicates that insulinemic levels seem to influence the ovarian response to FSH. This is suggested by the enhanced recruitment of antral follicles and the higher rate of ovarian growth observed in the hyperinsulinemic group during treatment. As a consequence of this exaggerated follicular development, a higher prevalence of OHSS was found in these patients.
However, data of our paper suggest that insulin levels do not affect the gonadotropin dose necessary for follicular maturation, because the dose/BMI ratio was similar in both groups.
Considering the higher prevalence of obesity among hyperinsulinemic patients, we separately analyzed the outcome of FSH treatment in obese and lean patients in the normoinsulinemic and the hyperinsulinemic groups.
It is interesting to note that, as expected, the rate of increase of ovarian dimensions observed in normoinsulinemic obese subjects is less than that achieved in normoinsulinemic lean subjects. On the other hand, in hyperinsulinemic patients, the presence of obesity failed to influence the ovarian response to FSH. This observation seems to minimize the importance of body weight as a factor capable of influencing the ovarian response to FSH in such subjects. Therefore, it may be suggested that insulin increases ovarian sensitivity to FSH. On the other hand, the higher FAI value apparently did not affect the follicular response to FSH.
The pregnancy rate seems to be negatively influenced by the high insulin circulating levels, but the relatively small number of pregnancies achieved does not allow for any conclusive observation regarding the fertility of hyperinsulinemic subjects. The incidence of spontaneous abortion is lower with respect to literature data for both treated and untreated patients. The high incidence of OHSS and the relatively small number of observations may account for this discrepancy. It is interesting to note that E2 production and E2/A ratio during ovarian stimulation were higher in hyperinsulinemic PCOS subjects, whereas no difference in production of A between the groups was seen.
It has been hypothesized that hyperinsulinemia increases androgen production by stimulating ovarian steroidogenesis (15). The human ovary possesses receptors for both insulin and insulin-like growth factor 1 (IGF-I); because there is cross-reactivity between these hormones and their receptors (16), hyperinsulinemia had been supposed to act both directly and via IGF-I receptors.
Erikson et al. (17), comparing the endocrine properties of granulosa cells from polycystic ovaries with those of normal granulosa cells from size-matched small follicles, found that polycystic ovarian cells showed increased sensitivity to FSH. In another experimental design by our group (18), PCOS granulosa cells showed a marked dose-dependent capacity for E2 production under in vitro insulin and IGF-I stimulation. Overall, these results suggest an intrinsic alteration of follicle steroidogenesis.
Therefore, our findings suggest that, in hyperinsulinemic patients, the increased response to exogenous FSH may be caused partly by the effect of insulin on the aromatase activity of granulosa cells. In fact, A may be considered the main substrate for ovarian aromatization. This observation associated with the presence of similar A levels in hyper- and normoinsulinemic subjects and increased values of E2 (and consequently, an increased E2/A ratio), support this hypothesis.
Furthermore, other data on PCOS granulosa cells showed that follicles arrested in the final stages of maturation are not necessarily atretic and, indeed, are hyperresponsive to gonadotropin stimulation. The author suggested that this may be caused by stimulation of aromatase activity in vivo (19). These data correspond to those of the present study, in which a larger number of small follicles were observed in hyperinsulinemic subjects with respect to normoinsulinemic ones.
In conclusion, the present study showed, for the first time, a significant difference in the ovarian response to FSH-induced ovulation in PCOS patients: hyperinsulinemic patients showed a greater endocrine and morphologic ovarian response. Such factors could lead to development of OHSS and could be associated with a decreased pregnancy rate.
Further studies are required for elucidating the mechanisms leading to these clinical observations.
Received April 2, 1996.
Revised June 26, 1996.
Revised September 24, 1996.
Accepted October 7, 1996.
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P < 0.05. B, Ovarian volume in normoinsulinemic
subjects. Normal weight vs. obese subjects. 
