1 Department of Obstetrics and Gynecology, Rush Medical College, Chicago, IL, 2 Repromedix Corporation, Woburn, MA and 3 Sher Institute and Millinova Laboratories, Chicago, IL, USA
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Abstract |
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Key words: age-related ovarian response/low responders/luteinization/ovarian antibodies/ovarian stimulation
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Introduction |
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Low gonadotrophin responses are usually differentiated from normal responses by the level of serum estradiol attained in response to 714 days of FSH stimulation during controlled ovarian stimulation. It has become customary to designate low responses as those <1000 mIU/ml estradiol (Scott, 1996; Ferraretti et al., 2000
). The number of ampoules of FSH required to attain an adequate response is also an indicator of low responses, although a specific cut-off value has not been defined. A reduced number of oocytes retrieved after hCG stimulation may also be associated with low responses (Dor et al., 1992
). However, poor responses associated with a low number of follicles recruited during controlled ovarian stimulation may represent a different clinical entity than low responses to gonadotrophin (Ferraretti et al., 2000
; Surrey and Schoolcraft, 2000
).
Different aetiologies have been described for low responses. For example, low responses are associated with ageing and low ovarian reserve (Kligman and Rosenwaks, 2001), reduced blood flow (Pellicer et al., 1994
), reduced aromatase activity (Hurst et al., 1992
), hormone resistance associated with specific FSH receptor polymorphism (Perez Mayorga et al., 2000
) and ovarian autoimmunity (Meyer and Lavy et al., 1990; Hovav et al., 1994
). Although ageing appears to explain a proportion of low responses, many low responders are young (Jenkins et al., 1991
; Kim 1995
; Hanoch et al., 1998
). Thus, it is likely that low responses represent multiple aetiologies.
In a previous study, we examined the possibility that differences in hormone response were due to differences in gonadotrophin receptor expression. We showed that FSH receptor expression was similar on granulosa cells of low and normal responders but LH receptor expression was slightly higher on cells of low responders (Thiruppathi et al., 2001). In addition, more of the granulosa cells from low responders had the large rounded, granular morphology of luteal cells (unpublished observation). Similarly, Whitman et al. observed that follicles of oocytes that failed to fertilize contained more luteinized cells (Whitman et al., 1988
, 1989
). These observations suggested that follicles of low responders might be more luteinized.
More advanced luteinization has been reported in follicles of older women during natural ovarian cycles (Klein et al., 1996a,b
,c
; Klein and Soules, 1998
). Based on serum measures, premature luteinization was observed in women with infertility (Ubaldi et al., 1996
; Lindheim et al., 1999
; Muttukrishna et al., 2000
). However, markers of luteinization have not been examined at the follicular level as a basis for altered responsiveness to gonadotrophin.
The objective of this study was to test the hypothesis that follicles of low responders were more luteinized than follicles of normal responders. Endocrine markers of luteinization such as progesterone, inhibin-A and vascular endothelial growth factor (VEGF) were assessed in follicular fluid and serum of low and normal responders at oocyte retrieval. A secondary objective was to determine if hormone profiles in the presence or absence of ovarian antibodies differed.
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Materials and methods |
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Ovarian stimulation was initiated by pituitary desensitization with GnRH during the midluteal phase of the preceding cycle. In the subsequent cycle, hMG, urofollitropin, or urofollitropin HP) was injected daily starting with an initial dose of 300 IU. Highly purified urinary FSH (Fertinex®; Serono, Norwell, MA, USA) was used for 70% of the patients, with the remaining FSH preparations distributed similarly among the study groups. Doses were adjusted based on follicle growth by ultrasound and serum estradiol monitored every other day. Ovulation was induced with 10 000 IU of hCG (Profasi®; Serono) after follicles were 17 mm in diameter and estradiol had increased over 8 days. The average estradiol response to gonadotrophin in the study cycle was 1341 ± 605 (range 5642714) and the average number of ampoules of FSH administered was 52 ± 28 (range 16120). Oocytes were obtained 34 h after hCG administration under transvaginal ultrasound guidance.
Measurement of hormones and antibodies in follicular fluid and serum
For each individual, the follicular fluid from three follicles, 1820 mm in diameter, was pooled in order to obtain adequate volume for multiple measures. The similarity of results from individual follicles and the pooled follicular fluid of six individuals was verified (Thiruppathi et al., 2001). The follicular fluid was centrifuged to remove cells and debris and frozen at 20°C until assay. Serum was separated from whole blood and stored at 20°C. Hormones were measured by immunoassay with commercial kits. Progesterone and estradiol were measured in serum and follicular fluid (Cayman Chemical, Ann Arbor, MI, USA) with detection limits of 10 and 9 pg/ml respectively. Androstenedione was measured in follicular fluid and dehydroepiandrosterone sulphate (DHEAS) were measured in serum (Diagnostic Systems Laboratories, Houston, TX, USA) with detection limits of 0.03 and 15 ng/ml respectively. VEGF was measured in follicular fluid by enzyme immunoassay (R&D Systems, Minneapolis, MN, USA). FSH was measured in follicular fluid (Genzyme, CA, USA). Inhibin-A and inhibin-B were measured with Serotec kits (Harlan, Raleigh, NC, USA). The sensitivity of the inhibin-A immunoassay was 10 pg/ml and of inhibin-B was 15 pg/ml. Hormone levels in follicular fluid were significantly higher than the range of the immunoassay standard curves. Therefore, samples were diluted with sample dilution buffer supplied by the manufacturer, until the levels were in the mid-range of the standard curve. The inter-assay and intra-assay coefficients of variation of all commercial assays were <10%.
Ovarian and oocyte antibodies in serum were determined as described previously (Luborsky et al., 1990; Luborsky and Pong, 2000
). In summary, anti-ovarian antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Ovaries were obtained from rats superovulated with pregnant mares serum gonadotrophin and HCG. The 10000g pellet of ovarian homogenates was used as antigen (0.1 µg/ 0.1 ml Tris buffer/well). Results with rat or human ovary were highly correlated (r = 0.95, P < 0.001) (Luborsky and Pong, 2000
). Anti-oocyte antibodies were detected in ELISA using homogenates of oocytes from mature follicles of rat ovaries. Oocytes were aspirated from follicles at the ovary surface, incubated in 0.5% collagenase (Sigma, St Louis, MO, USA) and 0.01% DNAase (Sigma) in PBS (30 min, 37°C) and separated from cells by gradient centrifugation in 3 ml of Hypaque (Sigma) (400 g, 30 min). After washing, oocytes were homogenized in batches of 100 in a 1 ml microfuge tube with a hand-held micro-pestle. Oocytes were added to ELISA plates at a concentration of
1 oocyte/0.1 ml Tris buffer/well. Results with rat or human oocytes were correlated (r = 0.85, P < 0.001).
Sera were tested at a dilution of 1:100 and follicular fluid at a dilution of 1:10. Negative control sera from normally cycling women (n = 10) and a positive control were included in each assay. For follicular fluid, four control samples from women with male factor infertility were used as negative controls. The captured autoantibodies were identified by a goat antihuman IgGalkaline phosphatase conjugate (FAB specific; Sigma). Bound alkaline phosphatase was reacted with substrate (AP substrate; Sigma) and the product read at 405 nm (Thermomax; Molecular Devices, Sunnyvale, CA, USA).
Data and statistical analysis
Statistical differences between normal and low responders and among the response groups were assessed with the MannWhitney U-test. Pearsons correlation coefficient (r) was calculated to evaluate associations between variables. P < 0.05 was considered significant.
For detection of ovarian and oocyte auto antibodies, samples were assessed in duplicate against four negative control samples and a positive control. Inter-assay and intra-assay coefficients of variation for specific optical density signals were <15% and <10% respectively. In this study, individuals positive for one or both antibodies were considered positive and results are referred to in the text as ovarian antibody positive for simplicity. Although ovarian antibodies were correlated in follicular fluid and serum (r = 0.4, P = 0.035), the serum test results were used to differentiate positive and negative low responders. Results were compared by 2-analysis.
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Results |
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The correlation of gonadotrophin response and indicators of luteinization were also assessed without separating the study group into low and normal responses. Response to gonadotrophin was negatively correlated with age (r = 0.4, P = 0.04), follicular fluid FSH levels (r = 0.5, P = 0.003) and the inhibin-A/inhibin-B ratio (r = 0.5, P = 0.013), but was not correlated with follicular fluid estradiol, progesterone or the progesterone/estradiol ratio. The lack of correlation of the progesterone/estradiol ratio with gonadotrophin response reflects a difference in the distribution of progesterone levels for normal and low responders. As seen in Figure 3, progesterone was correlated with gonadotrophin response in normal responders (r = 0.72, P = 0.006) but was not correlated among low responders (r = 0.05, P = 0.4, not significant). When ovarian antibody positive individuals were excluded, the correlation of response with progesterone (r = 0.4, P = 0.055) and inhibin-B (r = 0.4, P = 0.067) approached significance.
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Hormones in serum
Serum levels of hormones were measured as an indicator of the overall activity of follicles in the ovary. Serum inhibin-A and inhibin-B (Table IV) and peak estradiol (day of hCG) (Table I
) were significantly lower in low responders than normal responders. Estradiol (day of oocyte retrieval), the inhibin-A/inhibin-B ratio, progesterone and the progesterone/estradiol ratio were not significantly different between low and normal responders. DHEAS was measured as an indicator of somatic ageing and of adrenal steroid biosynthesis and differed significantly between normal and low responders (Table IV
). However, DHEAS in low responders without ovarian antibodies was significantly higher than DHEAS in low responders with ovarian antibodies and in normal responders (Figure 4
).
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The number of FSH ampoules was negatively correlated with serum inhibin-B (r = 0.5, P = 0.01) regardless of the inclusion of ovarian antibody positive individuals and was not correlated with serum levels of other hormones.
Relationship between serum and follicular fluid hormone levels
With a few exceptions, serum hormone levels did not reflect patterns of follicular fluid hormones regardless of the inclusion or exclusion of ovarian antibody positive individuals in the analysis. For example, progesterone in serum and follicular fluid were not correlated (P = 0.08, not significant). When ovarian antibody positive individuals were excluded, serum estradiol (day of oocyte retrieval) (r = 0.53, P = 0.02) and serum inhibin-A (r = 0.46, P = 0.04) were negatively correlated with the follicular fluid inhibin-A/inhibin-B ratio. In other words, in the absence of ovarian antibodies, the lower the serum estradiol and the higher the inhibin-A/inhibin-B ratio the more likely that individual maturing follicles would exhibit accelerated luteinization.
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Discussion |
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Only low responders without ovarian antibodies showed a clear trend towards accelerated luteinization and higher age. Low responders with ovarian antibodies tended to be similar to normal responders with respect to age and hormone profiles, rather than to low responders without ovarian antibodies. Also, hormone levels and oocyte parameters varied more in low responders with ovarian antibodies.
The higher VEGF observed in this study is consistent with previous reports that higher levels of VEGF were associated with lower peak estradiol levels, lower number of oocytes retrieved, higher age and higher number of FSH ampoules required (Friedman et al., 1997, 1998
). In addition, Neulen et al. showed that the ratio of VEGF/soluble VEGF receptor (sVEGFR-1) was inversely related to the number of oocytes retrieved, suggesting that decreased bioavailability of VEGF is associated with poor responses defined by oocyte yields (Neulen et al., 2001
). VEGF is required for corpus luteum formation and increases with progressive luteinization and vascular growth (Ferrara and Davis-Smyth, 1997
; Ferrara et al., 1998
). VEGF production in luteinizing granulosa cells is stimulated by FSH and LH (Christenson and Stouffer 1997
; Laitinen et al., 1997
; Geva and Jaffe, 2000
; Stouffer et al., 2001
). Likewise, VEGF was correlated with FSH in this study. However, since doses of FSH are not equivalent, it is not clear if the higher levels of VEGF are a consequence of higher doses of FSH, if luteinization is enhanced by a slightly higher content of LH in the FSH preparation (Filicori et al., 2001
) or if low responders have reduced control of the rate of follicle growth and differentiation (Eppig, 2001
).
Serum hormone levels did not reflect the hormone profiles of mature follicles. Instead, serum hormone levels tended to be lower in low responders. Both low responders with and without ovarian antibodies had lower levels of ovarian hormones consistent with reduced ovarian reserve that is thought to precede ovarian failure. In serum, the hormone levels reflect the additive activity of follicles of different maturation stages. Increasing estradiol in response to high doses of FSH may be due to increases within smaller follicles or to recruitment of additional, less mature, follicles. The trend to lower serum levels of ovarian hormones in low responders and in older women is consistent with other studies (Scott, 1996; Hall et al., 1999
; Seifer et al., 1999
). For example, Seifer et al. reported that age was associated with declining ovarian reserve, decreased serum inhibin-B, increased requirement for FSH, lower success rate and fewer oocytes retrieved (Seifer et al., 1999
).
We did not observe a difference in serum progesterone between normal and low responders. Other reports describing the relationship of serum progesterone and responses to gonadotrophin are inconsistent. Lindheim et al. found that low serum progesterone responses to hCG were associated with poor estradiol responders and suggested that this reflected inadequate steroidogenesis (Lindheim et al., 1999). Women with premature luteinization, defined by serum progesterone >1.1 ng/ml on the day of hCG administration, required a higher number of FSH ampoules to attain an adequate estradiol response (Ubaldi et al., 1996
). Likewise, premature luteinization, defined by a serum progesterone/estradiol ratio >1 on the day of hCG administration, was associated with low estradiol responses and ovarian reserve (Younis et al., 2001
). Follicular fluid hormone levels were not measured in these studies, and thus there is no information on the activity of maturing follicles. Differences in conclusions among studies may reflect different types of information obtained by assessing serum or follicular fluid, differences in definition of low and normal responses, and differences in study design and the day of serum sampling.
Thus, it appears that rather than reduced responsiveness, follicles of older low responders are maturing and differentiating at a faster rate than those of normal responders. The results are consistent with previous reports of accelerated luteinization of follicles in non-stimulated ovarian cycles of older women (Klein et al., 1996a,b
; Klein and Soules, 1998
). Greater luteinization could be due to higher levels of FSH, higher levels of LH associated with higher doses of FSH, enhanced responses to low levels of LH as cells acquire LH receptors (Filicori et al., 1999
, 2001
), or to a reduced ability of oocytes to inhibit luteinization (Vanderhyden and Macdonald, 1998
) prior to oocyte retrieval.
The results are consistent with heterogeneous aetiologies of low responses. Tanbo et al. reported two types of low responses based on clomiphene citrate responses (Tanbo et al., 1990). Hanoch et al. also differentiated low responses by age and noted that young low responders had better pregancy rates (Hanoch et al., 1998
). Low estradiol responses were also differentiated by day 3 LH (Noci et al., 1998
). FSH receptor polymorphism at position 680 was associated with different requirements for FSH during stimulation, suggesting partial hormone resistance in some women (Perez Mayorga et al., 2000
). Low responses have also been associated with decreased blood flow measured by Doppler ultrasonograpy (Pellicer et al., 1994
). We showed that some low responders had ovarian antibodies (Meyer et al., 1990
). In this study, we found that low responders differentiated by the presence or absence of ovarian antibodies differed in age and hormone profiles. Because of the heterogeneity of low gonadotrophin responses, it has been suggested that pre-cycle screening to differentiate potential responses may improve treatment outcomes (Surrey and Schoolcraft, 2000
).
Although the primary objective of this research study was to obtain information on potential mechanisms of low responses to FSH, there are implications for clinical practice. The lack of correlation of FSH dose with peak serum estradiol, the correlation with follicular fluid markers of luteinization and the lack of difference in oocytes retrieved suggest that higher FSH doses are not required for older low responders. This is consistent with other studies (Tinkanen et al., 1999; Out et al., 2000
). Premature luteinization associated with ageing may result in over-ripe or poorer quality oocytes (Eichenlaub-Ritter, 1998
; Sauer, 1998
). It has been reported that monitoring inhibin-B in addition to estradiol to determine the timing of hCG improves prediction of a successful outcome compared with estradiol monitoring alone (Seki et al., 1997
). This would be supported by this study since serum inhibin-B was correlated with the follicular response to gonadotrophin. Since the rate of follicle growth is slower in response to recombinant FSH which lacks trace LH (Filicori et al., 1999
, 2001
), it has been suggested that outcomes for low responders may be improved by using recombinant FSH or a combination of recombinant FSH and controlled levels of trace LH, although treatments may need to be individualized (Pellicer et al., 1998
; Howles, 2000
; Surrey and Schoolcraft, 2000
; Fawzy et al., 2001
).
The significant differences among low responders with ovarian antibodies suggest that treatment responses to standard hormone therapy may be less predictable in low responders with ovarian antibodies. Thus, individuals with ovarian antibodies may benefit from closer pre-cycle screening and monitoring during treatment for markers of follicular growth. In some cases, hormone stimulation may overcome the autoimmune reaction and recruit some healthy follicles. However, there is evidence that treatment outcomes associated with ovarian autoimmunity are poor (Geva et al., 1999; Ivanova et al., 1999
; Horejsi et al., 2000
; Luborsky and Pong, 2000
). Treatment with low dose immunosuppression has been successful (Luborsky et al., 1990
; Geva et al., 1999
), but treatment protocols have not been standardized in controlled trials.
In summary, mature follicles of low responders appear to be more luteinized than follicles of normal responders. In the absence of ovarian antibodies, low responses are associated with higher chronological and somatic age and accelerated luteinization of mature follicles, rather than diminished responsiveness. However, the follicular environment of low responders with ovarian autoantibodies differs from low responders without autoantibodies, suggesting different mechanisms for low responses in the presence of ovarian autoimmunity. In addition, use of ovarian antibody testing provides an additional tool for predicting response to gonadotrophin and may contribute to more individualized regimens for controlled ovarian stimulation.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 4, 2002; accepted on May 23, 2002.