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Original Studies |
Department of Reproductive Science and Medicine, Division of Pediatrics, Obstetrics and Gynecology, Imperial College of Science, Technology, and Medicine, St. Mary’s Hospital (D.S.W., H.W., H.D.M., S.F.), London, United Kingdom W2 1PG; and the Department of Obstetrics and Gynecology, University of Malta, St. Luke’s Hospital (R.G., M.B.), Gwardamangia, Malta
Address all correspondence and requests for reprints to: Dr. Debbie Willis, Department of Obstetrics and Gynecology, Imperial College School of Medicine, St. Mary’s Hospital, London, United Kingdom W2 1PG. E-mail: d.willis{at}ic.ac.uk
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Abstract |
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Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA.
In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5–3.9) and P, 1.0 (0.3–2.5) in ovulatory women and E2, 1.4 (0.7–25.4) and P, 1.3 (0.3–7.0) in anovulatory women.
Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.
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Introduction |
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Obese women with polycystic ovaries are more likely to be anovulatory and experience menstrual disturbance, and are less likely to ovulate in response to clomiphene citrate or gonadotropin administration despite similar increases in FSH than their lean counterparts (6, 7, 8). Conversely, improvement in the insulin status of anovulatory women with PCOS by weight loss, accompanied by a reduction in insulin levels, or insulin-lowering drugs can lead to a resumption of ovulatory cycles and fertility in some patients (9, 10, 11, 12).
We have previously shown that despite peripheral insulin resistance in women with PCOS, the polycystic ovary remains responsive to insulin (in terms of steroidogenesis) and that insulin acts via its own receptor rather than the type I insulin-like growth factor receptor (13, 14). Preincubation with insulin sensitizes human granulosa cells to LH, greatly enhancing granulosa cell progesterone (P) production in response to LH stimulation (14). This suggests enhanced granulosa cell differentiation by insulin in vitro (14). We propose, therefore, that hyperinsulinemia in anovulatory women with PCOS may lead to premature maturation of granulosa cells in vivo.
If this hypothesis is correct, granulosa cells from anovulatory women with polycystic ovaries (anovPCO) may respond to LH at an earlier stage of development than granulosa cells from ovulatory women with polycystic ovaries (ovPCO) or normal ovaries (since hyperinsulinemia is associated with anovulation in women with polycystic ovaries).
Hence, the aims of this study were 1) to determine at what follicle size human granulosa cells from normal ovaries and polycystic ovaries respond to LH in terms of steroid production in vitro, and 2) to compare the response to LH in granulosa cells from anovulatory women with polycystic ovaries with that in ovulatory women with either normal or polycystic ovaries.
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Subjects and Methods |
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. No patient had been receiving
treatment for suppression or stimulation of the ovaries for at least 3
months before surgery. Approval for the use of ovarian tissue in this
study was obtained from Kensington, Chelsea, and Westminister Health
Authority ethical committees and the research ethics committee of the
Faculty of Medicine and Surgery of the University of Malta.
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The number and size range of follicles used in the study were: 25 follicles 13 mm or smaller and four preovulatory follicles from normal ovaries, 62 follicles 12 mm or smaller and two preovulatory follicles from ovPCO, and 35 follicles 8 mm or smaller from anovPCO.
Granulosa cell cultures
Granulosa cells were isolated and cultured as previously described (14). Briefly, granulosa cells from individual follicles greater than 4 mm were cultured separately, and follicles 4 mm or smaller from individual patients were pooled, except in the case of two 3.5-mm and five 4-mm follicles from which sufficient cells were obtained to be cultured separately. Where granulosa cell number permitted, cells were plated at 5 x 104 viable cells/well in 200 µL serum-free medium 199 supplemented with antibiotics (penicillin and streptomycin) and 200 mmol/L glutamine (Life Technologies, Paisley, Scotland), and experiments were performed in triplicate. In the majority of experiments, when insufficient cells were obtained to achieve this, we used a reduced plating density in duplicate or, if necessary, singleton wells.
The granulosa cells were incubated with LH (5 ng/mL) or, as a positive control, FSH (5 ng/mL) in the presence of 5 x 10-7 mol/L testosterone, as aromatase substrate, for 48 h. When the number of granulosa cells in a single follicle was sufficient (as in dominant or preovulatory follicles) additional doses of LH and FSH (0.625–10 ng/mL) were tested. FSH concentrations of 2.5 and 5 ng/mL were commonly used because in previous studies these were found to give maximal estradiol (E2) responses (16). Gonadotropin preparations were highly purified human pituitary FSH (<16 IU LH/mg) and LH (<7 IU FSH/mg) supplied by Dr. S. Lynch of Endocrine Services (Bidford on Avon, UK). At the end of the 48-h incubation, the medium was removed and stored at -20 C until measurement of E2 and P by RIA as previously described (14). Viable granulosa cell number was assessed using the trypan blue exclusion test after isolation and in some cases at the end of the incubation period using a commercially available kit (CellTiter 96 nonradioactive cell proliferation assay, Promega Corp., Southampton, UK).
Where experiments were conducted in triplicate wells, the results are shown as the mean and SE of these triplicates. In these experiments, if error bars are not visible, the errors are contained within the mean bar. When comparing the response of granulosa cells to gonadotropins in multiple patients, statistics were performed using Student’s t test, Wilcoxon signed rank test, and Mann-Whitney U test as appropriate. Significance was assumed when P < 0.05.
Follicular fluid steroid levels
Follicular fluid E2 and androstenedione concentrations were determined in some follicles to assess the health of the individual follicles. These were determined using the same RIAs as those employed for the determination of steroid levels in the culture medium and were previously described in detail (14, 17).
Serum hormone measurements
Serum LH and FSH levels were measured using a commercially available kit from Chelsea Kits, Division of Molecular Endocrinology, Hammersmith Hospital (London, UK) (17A ). Fasting serum insulin levels were determined by RIA as previously described (5). The assay used a polyclonal antibody to insulin that cross-reacts with proinsulin (100%) and C peptide (3.8%).
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Results |
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Normal ovary
The effect of gonadotropin treatment on E2 production
by granulosa cells isolated from a range of follicle sizes from 6 of
the 10 patients with normal ovaries is shown in Figs. 1 and 2.
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9 mm)
In granulosa cells isolated from small antral follicles (9 mm)
from normal ovaries and then cultured in vitro, 16 of 20
cultures responded to FSH. FSH, as expected, significantly stimulated
E2 [median (range) fold increase, 2.0 (0.7–3.4);
P < 0.002] and P accumulation [median (range) fold
increase, 2.5 (0.3–5); P < 0.003]. There was no
effect of LH treatment on either E2 or P production in
these granulosa cell cultures (E2, P >
0.9; P, P > 0.4). Figures 1
and 2 show the effect of
LH and FSH on E2 and the corresponding P accumulation,
respectively, in granulosa cell cultures from a range of follicle sizes
from women with normal ovaries. Figures 1i and 2i illustrate the data
obtained from granulosa cells pooled from 1.5-, 2-, and 3-mm follicles
(a); from a 5-mm follicle (b); and from a 7-mm follicle (c) all
isolated from the same patient with normal ovaries who was in the
midfollicular phase of her menstrual cycle. There was no effect of
gonadotropins on steroid levels in granulosa cell cultures from 1.5-,
2-, and 3-mm follicles. As expected, FSH augmented E2 (Fig. 1i) and P (Fig. 2i) accumulation in granulosa cells from the 5- and
7-mm follicles. As the 7-mm follicle was the dominant follicle, more
cells were obtained in which to study gonadotropin action, and FSH and
LH were tested at two concentrations (2.5 and 5 ng/mL). FSH
dose-dependently stimulated E2 and P production, whereas
there was no effect of LH on steroidogenesis at either concentration
tested (only data for the 5 ng/mL dose are shown in Figs. 1i and 2i).
There was no effect of LH on steroid production in granulosa cells from
7.5-, 8-, or 9-mm follicles (Figs. 1ii and 2ii, 1iii and 2iii, and 1iv
and 2iv, respectively) from normal ovaries, but as expected, FSH
enhanced steroid levels in these cell cultures.
Antral follicles (>9 mm)
Six follicles larger than 9 mm were obtained from normal ovaries
(one 10 mm, one 13 mm, and four preovulatory follicles). In granulosa
cell cultures from follicles 10 and 13 mm in diameter, both FSH and LH
augmented E2 (Fig. 1, v and vi, respectively) and P (Fig. 2, v and vi, respectively) accumulation. Results obtained for the
preovulatory follicles are shown below, together with those obtained
from preovulatory follicles from ovPCO.
Ovulatory polycystic ovaries
As expected, more follicles were obtained from ovPCO than from
normal ovaries; hence, a wider range of follicle sizes was available
for study. Granulosa cells from ovPCO had a similar response to
gonadotropins as granulosa cells from normal ovaries. Figure 3 illustrates the effects of LH and FSH
on E2 production in granulosa cells isolated from a range
of follicle sizes obtained from three ovulatory patients with
polycystic ovaries. The corresponding data for P accumulation in these
granulosa cell cultures are shown in Fig. 4. Figures 3i and 4i illustrate the
response to gonadotropins in granulosa cells from a range of follicle
sizes obtained from the same patient. It highlights the range of basal
and gonadotropin-stimulated steroid levels in granulosa cell cultures
from follicles isolated from the same patient.
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9 mm)In granulosa cells from follicles 9 mm or smaller from ovPCO, 42 of 47 responded to FSH with a significant increase in E2 [median (range) fold increase, 2.7 (0.8–25.0)] and P accumulation [median fold (range) increase, 3.7 (0.6–31.5)] above basal levels in the cell cultures (E2, P < 0.0001; P, P < 0.001). Although a granulosa cell response to LH was observed in two of these follicles, there was no significant effect of LH on steroidogenesis in granulosa cell cultures obtained from follicles 9 mm or smaller (E2, P > 0.8; P, P > 0.9).
Antral follicles (>9 mm)
Nine follicles larger than 9 mm were obtained from ovulatory
polycystic ovaries (three 9.5-mm, one 10-mm, three 12-mm, and two
preovulatory follicles). The granulosa cells from all three of the
9.5-mm follicles (obtained from different patients) significantly
responded to FSH and LH (Figs. 3ii and 4ii). In Figs. 3ii and 4ii, the
data were pooled from three individual experiments in which granulosa
cells were isolated from three 9.5-mm follicles obtained from different
patients to highlight the significance of granulosa cells acquiring the
ability to respond to LH once follicles reach 9.5 mm in diameter in
ovulatory women with polycystic ovaries. The only 10-mm follicle
obtained from an ovulatory woman with polycystic ovaries was presumed
to be atretic; it had few granulosa cells, associated with low basal
steroid production, and did not respond to either gonadotropin (data
not shown). This patient had been in the midluteal phase of her
menstrual cycle; therefore, her ovary would not have been expected to
contain a healthy 10-mm follicle.
The responses of granulosa cells from three 12-mm follicles obtained
from different patients were varied. One, from a patient in the late
follicular phase of her menstrual cycle, appeared to be a healthy
dominant 12-mm follicle. This follicle was considered healthy because
it was well vascularized and contained 1,000,000 granulosa cells, and
the follicular fluid was estrogen dominant (18, 19) (E2,
3.11 µmol/L; androstenedione, 0.8 µmol/L). In granulosa cell
cultures from this healthy dominant follicle, LH augmented
E2 and P accumulation (Figs. 3iii and 4iii,
respectively).
In the other two 12-mm follicles, FSH, but not LH, augmented E2 production. However, LH-stimulated P accumulation in one of these cultures. Although these two 12-mm follicles were the largest follicles obtained in each patient, they did not appear to be healthy dominant follicles; they contained relatively few granulosa cells (300,000 and 600,000), and the follicular fluid was androgen dominant in both follicles (E2, 0.03 and 0.02 µmol/L; androstenedione, 11 and 12 µmol/L, respectively).
Preovulatory follicles from N ovaries and ovPCO
In total, six preovulatory follicles were obtained for
investigation. In four of six follicles (three N and one ovPCO), FSH
and LH stimulated both E2 and P accumulation in the
granulosa cell cultures (Fig. 5, i and
ii). In the other two preovulatory follicles (one N and one ovPCO), FSH
and LH augmented granulosa cell P accumulation only and had no effect
on E2 accumulation above the already high basal
E2 levels [E2 (Fig. 5iii) and P (Fig. 5iv)].
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Ovarian tissue was obtained from three anovulatory women with
PCOS. Two of the patients were hyperinsulinemic, as judged by the
fasting serum insulin concentration (Table 1) (5). Figures 6 and 7
illustrate the effects of LH and FSH on steroid production in granulosa
cells obtained from a range of individual follicle sizes from two of
the three anovulatory patients with polycystic ovaries. Figures 6i and 7i show data from follicles 4 mm or smaller from one patient, whereas
Figs. 6ii and 7ii show results from follicles larger than 4 mm from a
second patient. As seen previously in N and ovPCO groups, there was a
wide range of basal and gonadotropin-stimulated steroid levels among
follicles from the same patient. Although some of the follicles were
presumed to be atretic (with low granulosa cell number and low basal
steroid levels that were not augmented by FSH or LH; Fig. 6, ib, iic,
and iid), the proportion of atretic follicles in the anovPCO was
similar to that in N ovaries, in that 22 of the 28 granulosa cell
cultures responded to FSH. The median (range) fold increases in
E2 and P accumulation above the control values in response
to FSH were 1.4 (0.3–22.9) and 1.5 (0.3–25.2), respectively.
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). However, in marked contrast to the results obtained in
ovaries from ovulatory subjects, granulosa cells isolated from 4- to
8-mm follicles from anovulatory women with polycystic ovaries that
responded to FSH also responded to LH (Figs. 6 and 7). Analysis of
responses in all follicles 8 mm from anovPCO showed that FSH and LH
significantly stimulated E2 (FSH, P <
0.01; LH, P < 0.03) and P (FSH, P <
0.0001; LH, P < 0.02) accumulation.
Interestingly, granulosa cells isolated from the four largest follicles
from one anovulatory patient (two 7.5-mm follicles and both of her 8-mm
follicles) had a response to gonadotropin stimulation similar to that
seen in two of the preovulatory follicles described above. There was no
effect of LH or FSH on E2 production (Fig. 6ii, h–k), but
both gonadotropins augmented P accumulation in these same cultures
(Fig. 7ii, h–k, respectively), further suggesting that granulosa cells
in anovPCO may be prematurely advanced in their development.
Comparison of the response to LH in granulosa cells from ovulatory and anovulatory women
The effect of LH on granulosa cell steroidogenesis in ovulatory women (normal and polycystic ovaries) was compared to that in anovulatory women (with polycystic ovaries) in all FSH-responsive granulosa cell cultures from follicles 8 mm or smaller (as the maximum follicle size obtained from anovPCO was 8 mm in this study). The groups were matched for follicle size.
The responses to LH (expressed in terms of fold increase in steroid
production) were pooled from the results in all ovulatory women
(whether normal ovaries or ovPCO) and compared to the responses to LH
in granulosa cell cultures from anovPCO. The median and range fold
increases in E2 and P production in response to LH (5
ng/mL) are shown in Table 2. Granulosa
cells from follicles 8 mm or smaller from anovPCO were significantly
more responsive to LH than granulosa cells from size-matched follicles
from ovulatory women with normal ovaries or polycystic ovaries
(E2, P < 0.0003; P, P <
0.03).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To our knowledge this is also the first study documenting the steroid responses to LH in individual follicles in the human ovary. A clear, developmentally regulated response to LH was observed in human granulosa cells, consistent with previous studies (20, 21, 22, 23, 24), but we also showed that granulosa cells were able to respond to LH in ovulatory subjects (normal and polycystic ovaries) once follicles reached 9.5/10 mm in diameter.
Marked differences in basal steroid production and the magnitude of response to gonadotropins among cultures of granulosa cells obtained from different follicles of the same ovary were observed. This finding was anticipated because individual follicles in the same ovary are likely to be at varying stages of development or atresia. This is an important concept in follicle selection. It is generally assumed that at the time of selection, only one follicle is selected in humans because that follicle is at a slightly more advanced stage of development than other follicles in its cohort (25). Despite the observed variations in basal steroid levels and in the responsiveness to gonadotropins within patients, we found that granulosa cells from anovPCO women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries. In anovulatory women with polycystic ovaries, granulosa cells from follicles as small as 4 mm responded to both FSH and LH. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from size-matched follicles 8 mm or smaller from ovulatory women with normal or polycystic ovaries. This adds force to the argument that granulosa cells in anovPCO are at a prematurely advanced stage of development.
We propose that raised insulin levels in anovulatory women with polycystic ovaries may be a major factor causing premature maturation of granulosa cells in vivo, as we have previously shown in vitro that insulin increases the ability of granulosa cells to respond to LH (14). Hillier has suggested that the follicle requires LH throughout its development, but if LH levels are raised above a certain threshold level, premature luteinization may be initiated (the so-called ceiling hypothesis) (26). LH levels that are inappropriately high for the stage of follicle development have been shown to be detrimental to subsequent follicle maturation and ovulation (26, 27, 28, 29). Premature exposure of granulosa cells to LH inhibits their proliferation, such that development of the dominant follicle is arrested (26, 30, 31, 32). Furthermore, in normal follicle development, once granulosa cells can respond to LH (i.e. gain LH receptors) at 9.5–10 mm, they only undergo two more cell divisions to reach the preovulatory, 20–25 mm stage (33). In the anovPCO, granulosa cells were responsive to LH in follicles as small as 4 mm in diameter. Therefore, if the cells can only undergo two more divisions, they would be expected to reach a maximum size of around 8–10 mm (33).
A balance between FSH and LH is required for follicle development and ovulation (26). In anovulatory women with polycystic ovaries, raised insulin levels may upset this balance by sensitizing granulosa cells to the effects of LH. Administration of clomiphene citrate and exogenous FSH in ovulation induction programs may then redress the balance in favor of FSH (either by increasing the FSH/LH ratio or reaching a threshold level of FSH). This is supported by studies in rat granulosa cells where a FSH/LH ratio of 2 or less increased E2 production but had no effect on proliferation. However, increasing the FSH level to a FSH/LH ratio of 3 or larger stimulated granulosa cell proliferation (34).
As with all biochemical markers of PCOS, there are exceptions to the general trend that anovulatory women with PCOS are hyperinsulinemic; some anovulatory women with PCOS have normal serum insulin levels (5). The cause of anovulation in these patients is therefore not easy to explain on the basis of insulin action on the follicle. Such patients may, however, have a raised serum LH level (35), and therefore, it is possible that there is an interaction between insulin and LH such that either a raised insulin (and normal LH) or a raised LH (and normal insulin) level can lead to premature arrest of follicle growth and thus anovulation. Hyperandrogenemia in women with PCOS may also exacerbate the effects of insulin and LH. Androgens augment FSH-induced granulosa cell cAMP production and differentiation (36, 37). Raised insulin, LH, and androgen concentrations may, therefore, all have a role in prematurely advancing granulosa cell differentiation.
With a heterogeneous syndrome such as PCOS, there may be several causes
of anovulation. The mechanism of anovulation proposed here may be
relevant to hyperinsulinemic women with polycystic ovaries. Other
mechanisms that have been suggested are inhibitors of aromatase or FSH,
i.e. 5
-androstane-3,17-dione or the insulin-like growth
factor-binding proteins (38, 39).
In conclusion, the data presented here suggest that granulosa cell differentiation is prematurely advanced in anovulatory women with PCOS. This may contribute to the mechanism of anovulation in these women. It is proposed that hyperinsulinemia, by increasing the response of granulosa cells to LH, amplifies the effects of LH (which may already be elevated in these women). Thus, the effect of LH on the maturing follicle is similar to that which, in the normal cycle, is exerted only at later stages of development. This may lead to premature differentiation of granulosa cells, as high levels of LH inhibit granulosa cell proliferation and promote granulosa cell steroidogenesis and differentiation (26, 31, 32).
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Footnotes |
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Received March 3, 1998.
Revised July 14, 1998.
Accepted July 24, 1998.
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References |
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activity and serum free testosterone
after reduction of insulin secretion in polycystic ovary syndrome. N Engl J Med. 335:617–623.This article has been cited by other articles:
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S. Jonard and D. Dewailly The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest Hum. Reprod. Update, March 1, 2004; 10(2): 107 - 117. [Abstract] [Full Text] [PDF]
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D. A. Dumesic, R. D. Schramm, E. Peterson, A. M. Paprocki, R. Zhou, and D. H. Abbott Impaired Developmental Competence of Oocytes in Adult Prenatally Androgenized Female Rhesus Monkeys Undergoing Gonadotropin Stimulation for in Vitro Fertilization J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1111 - 1119. [Abstract] [Full Text] [PDF]
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R. M. Koivunen, L. C. Morin-Papunen, A. Ruokonen, J. S. Tapanainen, and H. K. Martikainen Ovarian steroidogenic response to human chorionic gonadotrophin in obese women with polycystic ovary syndrome: effect of metformin Hum. Reprod., December 1, 2001; 16(12): 2546 - 2551. [Abstract] [Full Text] [PDF]
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N. Ledee-Bataille, G. Lapree-Delage, J.L. Taupin, S. Dubanchet, J. Taieb, J.F. Moreau, and G. Chaouat Follicular fluid concentration of leukaemia inhibitory factor is decreased among women with polycystic ovarian syndrome during assisted reproduction cycles Hum. Reprod., October 1, 2001; 16(10): 2073 - 2078. [Abstract] [Full Text] [PDF]
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 J.E. Fortune, G.M. Rivera, A.C.O. Evans, and A.M. Turzillo Differentiation of Dominant Versus Subordinate Follicles in Cattle Biol Reprod, September 1, 2001; 65(3): 648 - 654. [Abstract] [Full Text] [PDF]
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A. J. Zeleznik Follicle Selection in Primates: ""Many Are Called but Few Are Chosen"" Biol Reprod, September 1, 2001; 65(3): 655 - 659. [Abstract] [Full Text] [PDF]
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F. Clement, D. Monniaux, J. Stark, K. Hardy, J. C. Thalabard, S. Franks, and D. Claude Mathematical model of FSH-induced cAMP production in ovarian follicles Am J Physiol Endocrinol Metab, July 1, 2001; 281(1): E35 - E53. [Abstract] [Full Text] [PDF]
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M. W. Elting, J. Kwee, R. Schats, L. T. M. Rekers-Mombarg, and J. Schoemaker The Rise of Estradiol and Inhibin B after Acute Stimulation with Follicle-Stimulating Hormone Predict the Follicle Cohort Size in Women with Polycystic Ovary Syndrome, Regularly Menstruating Women with Polycystic Ovaries, and Regularly Menstruating Women with Normal Ovaries J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1589 - 1595. [Abstract] [Full Text]
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A. J. Jakimiuk, S. R. Weitsman, A. Navab, and D. A. Magoffin Luteinizing Hormone Receptor, Steroidogenesis Acute Regulatory Protein, and Steroidogenic Enzyme Messenger Ribonucleic Acids Are Overexpressed in Thecal and Granulosa Cells from Polycystic Ovaries J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1318 - 1323. [Abstract] [Full Text]
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C. K. Welt and A. L. Schneyer Differential Regulation of Inhibin B and Inhibin A by Follicle-Stimulating Hormone and Local Growth Factors in Human Granulosa Cells from Small Antral Follicles J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 330 - 336. [Abstract] [Full Text]
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D. P. Levy, J. M. Navarro, G. L. Schattman, O. K. Davis, and Z. Rosenwaks The role of LH in ovarian stimulation: Exogenous LH: let's design the future Hum. Reprod., November 1, 2000; 15(11): 2258 - 2265. [Abstract] [Full Text] [PDF]
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 L. Poretsky, N. A. Cataldo, Z. Rosenwaks, and L. C. Giudice The Insulin-Related Ovarian Regulatory System in Health and Disease Endocr. Rev., August 1, 1999; 20(4): 535 - 582. [Abstract] [Full Text] [PDF]
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 J. F. Strauss III and A. Dunaif Molecular Mysteries of Polycystic Ovary Syndrome Mol. Endocrinol., June 1, 1999; 13(6): 800 - 805. [Full Text]
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