Recombinant human follicle stimulating hormone versus human menopausal gonadotrophin induction: effects in mature follicle endocrinology

M.P. Teissier1,3, H. Chable2, S. Paulhac1 and Y. Aubard1

1 Centre d'Assistance Médicale à la Procréation, Service de Gynécologie–Obstétrique, Centre Hospitalier Universitaire de Limoges, Limoges, 2 Service de Biochimie et Biologie Moléculaire et Faculté de Médecine, Limoges, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To investigate follicular effects of recombinant human follicle stimulating hormone (rhFSH) induction on women with polycystic ovary syndrome (PCOS), steroid content was compared in mature follicles obtained using a long luteinizing hormone-releasing hormone agonist plus rhFSH or human menopausal gonadotrophin (HMG) in PCOS women and controls participating in an in-vitro fertilization programme. Follicular fluids (144 samples) were collected at oocyte retrieval by individual selective aspiration. Oocyte maturity and fecundability were assessed. Plasma and intrafollicular 17ß-oestradiol, progesterone, testosterone concentrations were assayed individually. No significant difference was seen in oocyte maturity and fecundability between PCOS and controls following rhFSH, or between PCOS rhFSH and HMG group. 17ß-oestradiol, testosterone and progesterone concentrations were lower in PCOS follicular fluid following rhFSH than HMG but the difference was not significant. Progesterone concentration, 17ß-oestradiol/progesterone, 17ß-oestradiol/testosterone were significantly different between the two induction groups, for PCOS fertilized oocyte follicles (P = 0.01, P < 0.05 and P < 0.05 respectively). Steroidogenic enzymatic activity seems to be regulated in healthy follicular cells in PCOS as well as in normal patients upon ovarian induction. Following rhFSH, higher PCOS follicular progesterone concentrations leading to a theoretically increased fecundability could suggest that recombinant FSH is a better inducer which needs to be confirmed.

Key words: follicular sex steroids/human menopausal gonadotrophin/human recombinant FSH/in-vitro fertilization/polycystic ovary syndrome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the development of human recombinant follicle stimulating hormone (rhFSH) for in-vitro fertilization (IVF), several studies have found that rhFSH is beneficial for women with normal endocrine profiles (Devroey et al., 1994Go; Fisch et al., 1995Go; Hedon et al., 1995Go). Comparisons of rhFSH with urinary pure FSH (Mitchell et al., 1996Go; Bergh et al., 1997Go; Bloechle et al., 1997Go) or human menopausal gonadotrophin (HMG) in ovarian induction (Duijkers et al., 1997Go) have been limited to patients with a normal ovulation endocrine profile. No previous reports exist concerning polycystic ovary syndrome (PCOS) and rhFSH induction during in-vitro fertilization (IVF). PCOS is an ovarian disease characterized by the failure of spontaneous follicular maturation, inhibition of ovarian oestradiol biosynthesis and hyperandrogenization (Yen et al., 1993Go). rhFSH induction may have potential effect(s) on follicular development or oocyte maturation in PCOS women undergoing IVF. Follicular fluid is the endocrine environment of the oocyte and differences in steroid composition may affect oocyte quality. The aim of this study was to investigate whether PCOS ovarian stimulation with rhFSH or HMG resulted in different steroid composition of follicular fluid compared to controls. To achieve this, the follicular fluid steroid composition from mature PCOS follicles was analysed, following rhFSH or HMG induction, and steroid concentrations were compared in PCOS follicles containing fertilized oocytes, following rhFSH or HMG induction, in order to determine any difference between the two treatments.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
This study included 33 women with PCOS and 39 women with normal ovaries undergoing IVF. All patients (22–35 years old) had either a tubal factor or an anovulatory infertility. Male partners had normal semen quality according to World Health Organization (WHO) criteria. Before entering the IVF programme, PCOS and normal ovulation patients (considered as controls) were classified according to menstrual history and paraclinical data. Hormonal analysis and ultrasound examination were performed during the early follicular phase (day 2 to day 5) of a spontaneous cycle in order to determine ovulation profiles. The criteria used were as follows.

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 (2–8 mm diameter) formations around the cortex (at least 15) and/or stromal hypertrophia (Franks, 1989Go).

PCOS was diagnosed when at least two abnormalities of these parameters were observed (Franks, 1989Go; Yen et al., 1993Go). 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 pituitary–ovarian 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 150–225 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, 1982Go) 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 oocyte–cumulus 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, 1988Go).

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); oocyte–cumulus 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, 1994Go). Serum and FF testosterone concentrations were determined with Diria-TestoK kit (Sorin Biomedica Diagnostics, Seluggia, Italy; sensitivity = 0.05 ng/ml) (Furuyama et al., 1975Go). 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., 1979Go). For these steroid assays, intra- and inter-assay coefficients of variation (CV) were 4.5–5%, 6.5–7.8% and 6.9–10.1% 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 Mann–Witney's U-test. Linear correlation analysis (Pearson rank) was used to correlate the paramaters tested. Contingency analyses by least square ({chi}2) or Fisher's test, were performed to compare qualitative data. Significance was assumed at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Serum hormone concentrations in response to IVF stimulation (Table IGo)
In the rhFSH group, serum 17ß-oestradiol, progesterone and LH concentrations were lower in PCOS patients but not significantly different between PCOS and controls. Testosterone concentration was higher in PCOS serum than in controls (P = 0.0001). In the HMG group, serum 17ß-oestradiol, progesterone and LH concentrations tended to be higher in PCOS patients without any significant difference from control concentrations. Serum testosterone concentrations were also higher in PCOS than in controls (P = 0.0001).


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Table I. Comparison of serum steroid concentrations between PCOS and normal women following recombinant rhFSH or HMG ovarian induction (U-test)
 
However, statistical differences were seen for 17ß-oestradiol, testosterone and LH serum concentrations, which were lower in the PCOS rhFSH than in the PCOS HMG group (P < 0.05, P = 0.008 and P = 0.003 respectively).

Steroid concentrations in follicular fluids
Comparative analysis between follicular concentrations of mature follicles (Table IIGo)
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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Table II. Comparisons of steroid concentrations and ratios measured from mature follicles following the two treatments (U-test)
 
Relationship between induction treatment and oocyte maturity or fertilization
In the rhFSH group the number of mature oocytes did not significantly differ between PCOS (n = 25) and control groups (n = 35) ({chi}2 = 0.0001, P = 0.98). Among these 60 oocytes, 44 of them were fertilized: 21 were collected from PCOS and 23 from controls. No significant difference was seen for the fertilization rate between PCOS and controls (Fisher's test, corrected {chi}2 = 2.72, P = 0.058) (data not shown).

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) ({chi}2 = 0.001, P = 0.91).

Effects of steroid concentrations on oocyte fertilization
Results are shown in Tables IIIGo and IV.Go In Table IIIGo, steroid concentrations are shown for mature follicles containing fertilized oocytes and for unfertilized oocytes from PCOS patients.


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Table III. Comparison of steroid concentrations between PCOS mature follicles according to oocyte fecundability (U-test)
 

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Table IV. Comparison of steroid concentrations between PCOS follicles containing fertilized oocytes following the two treatments (U-test)
 
In the rhFSH group 17ß-oestradiol concentrations were higher in follicles that contained fertilized oocytes than in unfertilized oocytes (not significant). Testosterone concentrations were lower in fertilized oocyte FF than in unfertilized oocytes FF (P = 0.01). Progesterone concentrations were higher in fertilized oocyte FF than in unfertilized oocyte (P < 0.001). Both steroid ratios 17ß-oestradiol/progesterone, 17ß-oestradiol/testosterone differed significantly between PCOS follicles containing fertilized and unfertilized oocytes (P = 0.01 and P = 0.007 respectively) (Table IIIGo).

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 IIIGo).

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 IIIGo).

Table IVGo 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 IIIGo). 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 {chi}2 test, emphasized that oocyte maturity was correlated with fertilization rate ({chi}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).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study measuring in-vivo follicular 17ß-oestradiol, testosterone, progesterone concentrations was conducted to evaluate a new protocol of ovulation induction using recombinant FSH in PCOS, during an IVF programme.

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., 1998Go). 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., 1992Go; Mason et al., 1994Go; Agarwal et al., 1996Go). 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., 1980Go; Pache et al., 1992Go). 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., 1992Go). 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., 1992Go).

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., 1997Go). LH, plus HCG, increase follicular androgen production in the HMG group regardless of ovary status. Moreover, LH enhances aromatase activity (McNatty et al., 1980Go) 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., 1998Go). 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 IGo).

Concerning fertilization rate, the results present here of follicular oestrogen content are in agreement with those of earlier studies (Franchimont et al., 1989Go; Hartshorne, 1989Go; Branisteanu et al., 1997Go; Mantzavinos et al., 1997Go; Jacob et al., 1998Go). 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., 1979Go; Mantzavinos et al., 1997Go). 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 IIIGo). 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., 1994Go). 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 IVGo). This result is comparable with those of several studies (Carson et al., 1982Go; Gidley-Baird et al., 1986Go; Hartshorne, 1989Go; Enien et al., 1995Go) but opposed to those of others (Franchimont et al., 1989Go; Andersen, 1993Go). 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., 1996Go). 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 IIIGo). 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., 1994Go). 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., 1995Go).

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.


    Acknowledgments
 
We are grateful to Prof. M.Rigaud for his help and to Dr J.Cook for assistance in the preparation of the manuscript; also to Dr P.Piver and Dr A.M.Chinchilla for their participation in follicular fluid collection. This work was supported by a grant from Serono Laboratories Paris, France.


    Notes
 
3 To whom correspondence should be addressed at: Centre d'AMP, Service de Gynécologie–Obstétrique, CHU Dupuytren, Avenue Martin Luther King, 87042 Limoges cedex, France Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 22, 1999; accepted on May 27, 1999.