1 Centre d'Assistance Médicale à la Procréation, Service de GynécologieObstétrique, Centre Hospitalier Universitaire de Limoges, Limoges, 2 Service de Biochimie et Biologie Moléculaire et Faculté de Médecine, Limoges, France
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Abstract |
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Key words: follicular sex steroids/human menopausal gonadotrophin/human recombinant FSH/in-vitro fertilization/polycystic ovary syndrome
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Introduction |
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Materials and methods |
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Clinical data: Spontaneous menstrual cycle duration, presence of ovulation, dysovulation or anovulation on thermic curves (at least three), cutaneous hyperandrogenism signs, body mass index [BMI: weight (kg)/height2 (cm)] were noted.
Hormonal parameters: Luteinizing hormone (LH)/FSH ratio, prolactinaemia, plasma concentrations of testosterone, dehydroepiandrostenedione sulphate (DHEA-S) and 17ß-oestradiol.
Ultrasound pelvic examination: ovary size (normal volume <8 ml); presence of micropolycystic (28 mm diameter) formations around the cortex (at least 15) and/or stromal hypertrophia (Franks, 1989).
PCOS was diagnosed when at least two abnormalities of these parameters were observed (Franks, 1989; Yen et al., 1993
). Normal patients had none of these pathological criteria.
IVF protocol
All patients started, for their first cycle of IVF, a long gonadotrophin-releasing hormone agonist (GnRHa) protocol. They were all treated from day 1 of the menstrual cycle, until human chorionic gonadotrophin (HCG) administration, by 0.1 mg/day of D-Trp6 analogue (Decapeptyl 0.1®; Ipsen Biotech Laboratories, France). When pituitaryovarian axis down-regulation was achieved two induction groups were established. Forty-eight women (22 PCOS and 26 controls) were treated with rhFSH (Gonal F®; Serono Laboratories, Boulogne, France) and 24 patients (11 PCOS; 13 controls) were treated with HMG (Neopergonal®). In each group of ovarian induction, an initial dose of 150225 IU of FSH/day was administered i.m. This daily dose of FSH was maintained or increased in both groups, until adequate serum 17ß-oestradiol response was attained in agreement with ultrasound follicular growth monitoring. Then 10 000 IU HCG (5000 IU; Organon, Puteaux, France) were given when at least five follicles were present with an average follicular diameter >16 mm.
IVF procedures
Collection techniques and IVF
Oocytes and follicular fluids (FF) were individually collected for IVF by ultrasonographically guided vaginal puncture, 35 h after HCG administration. For each woman, two FF samples were obtained by a separate aspiration. Each sample was collected using a sterile syringe without culture medium. Follicular fluids selected were free of blood and contained only one morphologically normal oocyte, which was rapidly inseminated and cultured. All oocytes were inseminated in culture media containing 100 000 motile spermatozoa/ml. Embryological procedures were performed as previously described (Edwards and Purdy, 1982) but no bovine serum was added to culture media (Ferticult, J.C.D. SA, Gauville, France).
At the time of IVF puncture, blood samples (72 patients) were collected for determination of serum 17ß-oestradiol, LH, progesterone and testosterone concentrations.
Follicles and oocytecumulus complex classification
Subgroups of follicles were defined according to oocyte maturity. This maturity was studied at two different times: after retrieval and after fertilization (Veek, 1988).
After retrieval, the nuclear maturation of oocytes was used for grading. Oocytes were considered as mature (M) when the first polar body was extruded and as immature (IM) when the germinal vesicle and the first polar body were absent. Follicles were separated into the following functional classes: mature follicles (M-F) and immature follicles (IM-F) according to oocyte maturity.
After fertilization, mature oocytes were divided into two groups: fertilized oocytes (2-pronucleate oocytes with two polar bodies) and unfertilized oocytes which failed to divide.
Finally for the comparative study, FF was separated according to the following criteria: induction treatment (rhFSH or HMG); oocytecumulus complex maturity and fertilization capacity and ovulation group (PCOS or normal ovary patients).
Assays
Steroid measurement
Radioimmunoassay kits were used for all steroid measurements in serum and follicular fluid samples. The reproducibility and validity of the kits for FF assays were previously controlled (data not shown). Samples were diluted with pooled plasma obtained from men (n = 10) with undetectable 17ß-oestradiol and progesterone concentrations. All assays were performed at least twice. The mean was used for statistical analysis.
Serum and FF 17ß-oestradiol concentrations were determined with Coat-a-Count Estradiol kit (Berhing, Diagnostic Products Corporation, Los Angeles, CA, USA). The assay sensitivity was 8 pg/ml (Adashi, 1994). Serum and FF testosterone concentrations were determined with Diria-TestoK kit (Sorin Biomedica Diagnostics, Seluggia, Italy; sensitivity = 0.05 ng/ml) (Furuyama et al., 1975
). Serum and FF progesterone concentrations were determined using the Gamma Coat [125I]Progesterone CA 1724 kit (Incstar Corporation, Stillwater, MN, USA) with assay sensitivity of 0.11 ng/ml (March et al., 1979
). For these steroid assays, intra- and inter-assay coefficients of variation (CV) were 4.55%, 6.57.8% and 6.910.1% respectively.
Serum LH was measured using radioimmunoassay gnost hLH kit (Cis Bio-international kit, Oris, France). The sensitivity was 0.15 mIU/ml, intra-assay and inter-assay CV were 5% and 7% respectively.
Statistical analysis
Steroid concentrations were expressed as mean ± SEM. Comparisons between different FF classes were made using MannWitney's U-test. Linear correlation analysis (Pearson rank) was used to correlate the paramaters tested. Contingency analyses by least square (2) or Fisher's test, were performed to compare qualitative data. Significance was assumed at P < 0.05.
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Results |
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Steroid concentrations in follicular fluids
Comparative analysis between follicular concentrations of mature follicles (Table II)
17ß-Oestradiol, testosterone and progesterone concentrations in mature follicles tended to be lower in PCOS under rhFSH than in group HMG, but differences were not significant. The same tendency was observed in the control group. Comparative analysis of steroid ratios (17ß-oestradiol/progesterone, 17ß-oestradiol/testosterone) revealed some differences between mature follicles induced by both treatments. In PCOS, no significant difference existed for 17ß-oestradiol/progesterone ratio obtained after rhFSH or HMG induction. Similar results were seen in controls. 17ß-Oestradiol/testosterone was significantly higher in PCOS follicles from HMG group than from rhFSH (P = 0.003). In normal patients, follicular 17ß-oestradiol/testosterone ratio was higher in the rhFSH group than in HMG (P = 0.01).
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In the HMG group the same results were found, without any significant difference between PCOS and controls.
Finally, no difference existed between fertilization rate of PCOS oocytes from the rhFSH group (21 fertilized/25 mature) and from the HMG group (15/19) (2 = 0.001, P = 0.91).
Effects of steroid concentrations on oocyte fertilization
Results are shown in Tables III and IV.
In Table III
, steroid concentrations are shown for mature follicles containing fertilized oocytes and for unfertilized oocytes from PCOS patients.
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In the HMG group similar results were obtained but FF testosterone content did not differ between PCOS fertilized and unfertilized oocytes following HMG induction (Table III).
Finally the comparison of steroid concentrations from PCOS follicles containing fertilized oocytes, between rhFSH and HMG treatments, showed that 17ß-oestradiol and testosterone were lower in the rhFSH group, but not significantly different. Follicular progesterone concentration was higher in PCOS rhFSH follicles containing fertilized oocytes than in the HMG group (P < 0.01). 17ß-oestradiol/progesterone and 17ß-oestradiol/testosterone also differed significantly between follicular fluids according to their oocyte fecundability. 17ß-oestradiol/testosterone was higher in PCOS fertilized oocyte FF following HMG induction (Table III).
Table IV compares steroid concentrations from follicles containing fertilized oocytes from PCOS and control patients receiving rhFSH or HMG. No significant differences were seen between control and PCOS steroid concentrations in either group. However, comparison of steroid concentrations in PCOS patients showed that progesterone concentrations were significantly higher in patients undergoing rhFSH induction. 17ß-oestradiol/progesterone was lower in PCOS fertilized oocyte FF following both inductions and significantly lower from the rhFSH group (P = 0.04, Table III
). Similar results were seen when comparing control groups but this was not significant (data not shown).
Moreover, a significant correlation was reported between progesterone concentrations and oocyte maturity obtained from rhFSH and from HMG follicles induced (P = 0.002 and 0.05 respectively, data not shown). Contingency analysis, by 2 test, emphasized that oocyte maturity was correlated with fertilization rate (
2 = 0.13, P = 0.0003). This correlation was also observed in both PCOS and normal patients, without any difference between the two endocrine profiles (data not shown).
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Discussion |
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In mature PCOS follicles, 17ß-oestradiol concentrations tended to be significantly higher in the HMG than in rhFSH group (P = 0.06). In controls, the same tendency was observed. These findings are in agreement with those of another study (Jacob et al., 1998). Such a result could be explained by the use of rhFSH, a pure preparation completely devoid of LH activity. This result suggests that FSH may regulate aromatase activity by acting on the expression of this key enzymatic protein in normal or PCOS granulosa cells during ovarian follicular growth. This hypothesis is supported by earlier studies (Erickson et al., 1992
; Mason et al., 1994
; Agarwal et al., 1996
). Moreover in our study, using GnRHa induction protocol, serum LH is reduced in each patient group. The different composition of FSH and LH present in preparations used for induction could affect steroid concentrations in these growing follicles. High concentrations of exogenous LH could act on PCOS thecal cells resulting in higher 17ß-oestradiol concentrations from the HMG group. On the other hand, these findings underline that treatment with rhFSH, containing no LH, resulted in adequate 17ß-oestradiol, progesterone and androgen concentrations in antral fluid of ovarian follicles in women with PCOS and normal endocrine profiles. They also confirm those of Duijkers et al. (1997) in a study of patients with normal endocrine profile undergoing IVF induction with rhFSH. Our results concerning PCOS FF 17ß-oestradiol content are in agreement with previous studies (McNatty et al., 1980
; Pache et al., 1992
). It can be postulated that lower LH stimulation might result in a decrease in androgen production by thecal cells, and therefore lower 17ß-oestradiol production by granulosa cells resulting in lower 17ß-oestradiol follicular concentrations. A clinical study of a gonadotrophin-deficient woman induced by rhFSH supports this remark (Schoot et al., 1992
). rhFSH administration stimulated multiple follicular growth but with very low follicular steroid concentration and without concomitant increase in serum 17ß-oestradiol concentrations. FSH alone, without any LH, is not able to stimulate follicular steroid synthesis (Schoot et al., 1992
).
Follicular testosterone concentrations tended to be higher following HMG induction in PCOS as well as in controls. This result is in agreement with previous reports (Branisteanu et al., 1997). LH, plus HCG, increase follicular androgen production in the HMG group regardless of ovary status. Moreover, LH enhances aromatase activity (McNatty et al., 1980
) leading to testosterone conversion into 17ß-oestradiol which can explain why 17ß-oestradiol concentrations are higher in FF and in serum following HMG induction.
Progesterone concentrations were higher in PCOS mature follicles in HMG compared to rhFSH groups but not significantly. Follicular progesterone production was similar in PCOS and normal patient follicles under both conditions of ovarian treatment. This result strongly differs from those of a recent study which showed a decreased progesterone synthesis in PCOS cells (Doldi et al., 1998). Such a difference could be linked to the methodology used by these authors. They did not study the different functional follicle classes and performed the progesterone assay only in pooled size-matched follicles, probably mixing mature and immature follicles.
In the present study, in spite of pituitary suppression with GnRHa, low endogenous LH was sufficient to permit an adequate steroid production in mature follicles and their secretion into the blood following rhFSH induction in PCOS as well in controls (Table I).
Concerning fertilization rate, the results present here of follicular oestrogen content are in agreement with those of earlier studies (Franchimont et al., 1989; Hartshorne, 1989
; Branisteanu et al., 1997
; Mantzavinos et al., 1997
; Jacob et al., 1998
). No difference was observed in oestrogen concentrations between groups reflecting oocyte evolution in both IVF protocols as opposed to the follicular 17ß-oestradiol composition from a spontaneous cycle (McNatty et al., 1979
; Mantzavinos et al., 1997
). Moreover, higher testosterone concentrations were seen in unfertilized FF versus fertilized FF mature oocytes, in both rhFSH and HMG groups, but only differed significantly in the rhFSH group (Table III
). In accordance with Brzynski et al. (1995), it is suggested that excessive FF androgen concentrations may affect oocyte quality. Highest progesterone concentrations were observed in fertilized oocyte follicles, regardless of endocrine profile. Progesterone concentrations were significantly higher in rhFSH PCOS fertilized follicles versus HMG induced ones. Increased progesterone concentrations after the HCG/LH peak may be due to elevated androgen concentrations from the PCOS group which are converted after the HCG peak (Gilling-Smith et al., 1994
). It should be emphasized that following GnRHa plus rhFSH or HMG induction, healthy follicles from PCOS patients can produce adequate follicular progesterone concentrations. During induction, progesterone synthesis seems to be correlated with the functional status of follicles but not with the ovulation profile of the patient (data not shown). Significantly higher PCOS follicular progesterone and lower testosterone concentrations could suggest that rhFSH is a better inducer than HMG for PCOS patients.
Low 17ß-oestradiol/progesterone ratios are noted in PCOS fertilized oocyte FF from both groups (the lowest in rhFSH group) (Table IV). This result is comparable with those of several studies (Carson et al., 1982
; Gidley-Baird et al., 1986
; Hartshorne, 1989
; Enien et al., 1995
) but opposed to those of others (Franchimont et al., 1989
; Andersen, 1993
). It is suggested that the 17ß-oestradiol/progesterone ratio reflects follicle viability and may influence the fertilization stage of the oocyte. 17ß-oestradiol/testosterone ratios are significantly higher in fertilized follicles than in those which fail to divide. This ratio is linked to the aromatase activity which is more effective in healthy follicles (Kemper-Grenn et al., 1996
). Analysis of 17ß-oestradiol/testosterone ratios obtained from corresponding subgroups of follicles shows that 17ß-oestradiol/testosterone was highest in fertilized PCOS following HMG than rhFSH induction (Table III
). This finding suggests that the presence of exogenous LH in follicles (from HMG) may increase testosterone, and enhance aromatase activity, in mature follicles from PCOS as previously described from normal endocrine profile follicles (Yong et al., 1994
). Following induction, increased testosterone concentration probably leads to higher 17ß-oestradiol concentrations which are not necessarily correlated with a greater oocyte maturity when 17ß-oestradiol follicular hormone concentration is separately considered.
In summary, intrafollicular 17ß-oestradiol and progesterone concentrations do not strongly differ between PCOS and normal ovulation patients during GnRHa and rhFSH induction in an IVF protocol. Some differences exist in fertilized-oocyte follicle content (hormone concentration or ratio) in PCOS women depending upon the induction agent used. rhFSH seems to give a better result in PCOS than HMG, which may be due to lower follicular androgen concentrations and higher progesterone production leading to significant differences in steroid ratios (Enien et al., 1995).
This study emphasizes that: (i) with in-vivo rhFSH induction, luteinizing granulosa cells from PCOS healthy follicles can produce the same steroid endocrine environment as normal ovary cells and restore a comparable oocyte maturity; (ii) excess androgen is used as a substrate for oestrogen biosynthesis at this late stage of folliculogenesis and (iii) during an ovulation induction programme, when gonadotrophin concentrations are controlled, steroidogenic aromatase activity seems to be regulated in healthy follicular cells in PCOS as well as in normal patients leading to the same fertilization capacity of oocyte; (iv) androgen and progesterone concentrations seem to be more important than oestrogen concentration for oocyte fertilization.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 22, 1999; accepted on May 27, 1999.