B.C.Tarlatzis 1 ' 2 ' 3, G.Grimbizis 1 ' 2, F.Pournaropoulos 2, J.Bontis 2 * 4, S.Lagos 2, E.Spanos 2 and S.Mantalenakis 1 * 2

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1 Human Reproduction vol.10 no.10 pp , 1995 The prognostic value of basal luteinizing hormone:follicle-stimulating hormone ratio in the treatment of patients with polycystic ovarian syndrome by assisted reproduction techniques B.C.Tarlatzis 1 ' 2 ' 3, G.Grimbizis 1 ' 2, F.Pournaropoulos 2, J.Bontis 2 * 4, S.Lagos 2, E.Spanos 2 and S.Mantalenakis 1 * st and 4 2nd Department of Obstetrics and Gynecology, Aristotle University Thessaloniki and 2 Infertility and IVF Center 'Geniki Kliniki', Thessaloniki, Greece 3 To whom correspondence should be addressed at: Infertility and IVF Center 'Geniki Kliniki', 2 Gravias Street, Thessaloniki , Greece One of the main endocrinological disturbances in patients with polycystic ovarian syndrome (PCOS) is the increased baseline concentrations of luteinizing hormone (LH) and consequently a high LHrfollicle-stimulating hormone (FSH) ratio. The aim of this study was to assess the relationship between the baseline LH:FSH ratio with the stimulation response and the miscarriage risk in PCOS women stimulated for assisted reproduction techniques (ART) with and without gonadotrophin-releasing hormone analogue (GnRHa). Two groups of PCOS patients were analysed retrospectively. Group A (n = 20, 20 cycles) consisted of women stimulated with human menopausal gonadotrophin (HMG), and group B (n = 128, 162 cycles) comprised women stimulated with buserelin-long/hmg. LH and FSH concentrations were measured during the early follicular phase (days 4-6) in a preceding spontaneous or progestininduced cycle. The following parameters were assessed: number of follicles developed, number of oocytes obtained and percentage of mature oocytes, as well as number of abortions and live births. In group A, the baseline LH:FSH ratio was correlated inversely with the number of follicles developed (), the number of oocytes obtained (P < 0.05) and the percentage of mature oocytes (). In group B, no correlation was found between the LH:FSH ratio and the number of follicles and oocytes, because their numbers were relatively constant irrespective of the baseline LH:FSH ratio, but a significant inverse correlation was noted with the percentage of mature oocytes (P < 0.001). However, a comparison of the slopes of the curve indicated a better correlation between the LH:FSH ratio and the percentage of mature oocytes in group A than in group B (). These findings were also confirmed when patients were subdivided according to the LH:FSH ratio (<3 or 3=3). Furthermore, in women who miscarried, the mean LH:FSH ratio was significantly higher than in women having a live birth. In conclusion, in PCOS patients stimulated with HMG, a high basal LH:FSH ratio appears to have an adverse effect on the number of follicles and oocytes, as well as on oocyte maturity. On the other hand, Oxford University Press the administration of GnRHa in the long protocol seems to reverse this detrimental effect on follicle and oocyte development. Furthermore, a higher LH:FSH ratio seems to predict a greater possibility for miscarriage, despite the use of GnRHa. Key words: assisted reproduction techniques/gonadotrophinreleasing hormone analogues/luteinizing hormone/polycystic ovarian syndrome Introduction Infertility in patients with polycystic ovarian syndrome (PCOS) represents a common problem. Assisted reproduction techniques (ART) offer an alternative mode of therapy, with satisfactory results in cases of long-standing infertility previously resistant to other in-vivo treatment modalities. Such techniques also provide the opportunity to assess the fertilizing potential of the gametes (Tarlatzis and Grimbizis, 1993). One of the main endocrinological disturbances in patients with PCOS is the increased concentrations of luteinizing hormone (LH) and consequently a high LH:follicle-stimulating hormone (FSH) ratio. It seems that the raised concentrations of LH, as well as the premature LH surges, have an adverse effect on follicle and oocyte development, and are believed to be responsible for the relatively low pregnancy rates in this patient population (Homburg et al., 1988; Howies and MacNamee, 1990; Tarlatzis and Grimbizis, 1993). In PCOS patients stimulated for in-vitro fertilization (IVF) and embryo transfer, the percentage of immature oocytes is significantly higher compared with normo-ovulatory women (Dor et al., 1990). The use of gonadotrophin-releasing hormone analogues (GnRHa) in the long desensitization protocol seems to improve ART results, especially in women who have failed to conceive with other stimulation regimes, probably because of the suppression of endogenous LH and the prevention of premature LH surges (Tarlatzis and Grimbizis, 1993). However, once a pregnancy is achieved, the high miscarriage rate represents a serious clinical problem in this patient group (Tarlatzis and Grimbizis, 1993). To assess the effect of the basal endocrinological status on the stimulation response and the possible beneficial effect of GnRHa administration, we have analysed the correlation between the basal LH:FSH ratio and the number of follicles developed, the percentage of mature oocytes and the pregnancy outcome in cycles stimulated for IVF and embryo transfer or zygote intra-fallopian transfer (ZIFT) with human menopausal gonadotrophins (HMG) alone or in combination with the GnRHa buserelin in the long protocol. 2545

2 B.CTarlatzis et al. Materials and methods Definitions The diagnosis of PCOS was based mainly on ovarian morphology in an ultrasonographic assessment consisting of at least 10 cysts, 2-8 mm in diameter, arranged around a hyperechogenic central stroma (Adams et al., 1985; Balen et al., 1993). In addition, at least two of the following criteria were met: irregular menstruation (oligoamenorrhoea) with or without the clinical manifestations of obesity and hirsutism, an increased baseline LH:FSH ratio (>3), D 4 - androstenedione concentrations >2 ng/ml and/or testosterone concentrations >0.60 ng/ml, and in some cases laparoscopic appearance suggestive of PCOS. Patients and study design A total of 148 PCOS patients treated by FVF and embryo transfer or ZIFT were analysed retrospectively in 182 cycles. Baseline LH and FSH concentrations were measured in all patients, irrespective of the stimulation protocol used (HMG alone or buserelin-long/hmg), on days 4-6 of a preceding spontaneous cycle. In very few cases with extremely long amenorrhoea, a short-term progestin (Primolut N; Schering, Berlin, Germany) was used to induce menstruation. The indications for the treatment of PCOS patients with ART were: (i) long-standing infertility resistant to other in-vivo treatment modalities (at least four cycles), and (ii) the co-existence of another infertility factor, commonly tubal (Tarlatzis and Grimbizis, 1993). The patients were divided into two groups according to the stimulation protocol used. Group A consisted of 20 women (20 cycles) stimulated only with HMG (Humegon, Organon, Oss, The Netherlands; or Pergonal, Serono, Aubonne, Switzerland). HMG (225 IU) was initiated on day 3 of the cycle for 3 days, after a basic ultrasonographic assessment excluding the presence of cysts 5=10 mm in diameter. Thereafter, the dose was increased from cycle day 8 in a stepwise manner depending on the individual response, as monitored by both ultrasound scanning of the ovaries (Kretz Combison 320; Kretztechnik, Austria) and serum oestradiol determinations (Quant; Leeco Diagnostics Inc., Detroit, MI, USA). Human chorionic gonadotrophin (HCG; Pregnyl, Organon; or Profasi, Serono), IU i.m., was administered when two follicles of 2=16 mm in diameter were visualized and oestradiol concentration was >500 pg/ml. Patients from group A were analysed as a whole (linear regression analysis) and in two subgroups according to the LH:FSH values: those with LH:FSH values <3 (n = 11, 11 cycles) and those with LH:FSH values 3=3 (w = 9, 19 cycles). Group B consisted of 128 women (162 cycles) stimulated with buserelin (Suprefact; Hoechst AG, Frankfurt am Main, Germany) in the long desensitization protocol plus HMG. All patients received buserelin from day 1 of the cycle until pituitary desensitization, in a dose of 1 mg/day intranasally, divided into five puffs of 200 u.g each during the day. Hormonal and echographic monitoring was performed on day 15 to confirm oestradiol concentrations <50 pg/ml and the absence of growing follicles. Stimulation was then started with 150 IU HMG for 5 days. Thereafter the treatment was adapted to each patient's response (Tarlatzis et al., 1993). HCG was given when at least two follicles of 3*18 mm in diameter were present and the oestradiol concentration was > 1000 pg/ml. The treatment with GnRHa was then discontinued (Tarlatzis et al., 1993). As in group A, the patients were studied as a whole (linear regression analysis) and in two subgroups according to the LH:FSH ratio: those with a LH:FSH ratio <3 (n = 87, 113 cycles) and those with a LH:FSH ratio 3*3 (n = 41, 49 cycles). Furthermore, pregnant patients from group B (n = 34) were divided into two subgroups: subgroup Bl (n = 20), consisting of women who spontaneously miscarried in the first 2546 % D BuserefirMong/HMG * HMG LH/FSH ratio Figure 1. Linear regression analysis between the luteinizing number of follicles developed in polycystic ovarian syndrome menopausal gonadotrophin (HMG) alone (y = *, r , ) and (B, line b) buserelin-long/hmg (y = *, r = 0.05, P = 0.481). trimester, and subgroup B2 (n = 14), consisting of women with live births. The ages of patients in the two groups were similar (group A 28.2 ± 5.4 and group B 29.4 ± 6.2 years). Ultrasound-guided oocyte retrieval was performed 36 h after HCG administration in both groups. Oocyte classification and handling, as well as sperm preparation and embryo assessment, were performed as described previously (Laufer et al., 1984; Tarlatzis et al., 1986; Tarlatzis and DeCherney, 1987) by the same personnel and using the same criteria. Up to four zygotes or embryos were replaced. The luteal phase was supported by 1500 IU HCG on the day of transfer and on days 4, 8 and 12 thereafter if oestradiol concentrations were = 1800 pg/ml or with 50 mg natural progesterone (Lutorm; Elvipy SA, Athens, Greece) injections daily when oestradiol concentrations were >1800 pg/ml. Differences in the number of patients and cycles between the two groups were caused by the routine use of only the long protocol over the last 4 years in PCOS patients, whereas no other selection criteria were used for the allocation of the patients. Hormone analyses Serum concentrations of LH and FSH were measured by standard radioimmunoassays (Monobind, Costa Mesa, CA, USA). The interassay coefficient of variation (CV) for the individual analysis of both gonadotrophins was 4.2^1.5%, whereas the intra-assay CV was %. Statistical analysis Linear regression analysis was performed by examining the correlation between the basal LH:FSH ratio and the number of follicles developed, the number of oocytes recovered and the percentage of mature oocytes. Differences between the two groups were assessed by comparing the slopes of the curves obtained by linear regression analysis. Furthermore, the differences between the variables of the subgroups were compared by the unpaired Student's Mest or the % 2 test, as appropriate. Results In group A, the baseline LH:FSH ratio was inversely correlated with the number of follicles developed, e.g. the higher the LH:FSH ratio the lower the number of follicles (y = *, r = 0.45, ; Figure 1, line a). On the other hand, in group B no statistically significant correlation was found between the baseline LH:FSH ratio and the number of

3 LH:FSH in PCOS and ovarian response S 20 H LU/FSH ratio o Buserelin-tong/HMQ Figure 2. Linear regression analysis between the luteinizing number of oocytes retrieved in polycystic ovarian syndrome menopausal gonadotrophin (HMG) alone (y = *, r = 0.50, ) and (0, line b) buserelin-long/hmg (y = A:, r = 0.05, P = 0.575) % t I 80% H 5 60% 9 40%- a Buserelin-tong/HMG HMG LH/FSH ratio Figure 3. Linear regression analysis between the luteinizing percentage of mature oocytes in polycystic ovarian syndrome menopausal gonadotrophin (HMG) alone (y = *, r = 0.845, ) and (H, line b) buserelin-long/hmg (y = e" 2 x, r = 0.32, P = 0.001). follicles developed (y = *, r = 0.05, P = 0.481; Figure 1, line b), as the number of follicles was relatively constant independent of the LH:FSH ratio. However, a comparison of the curves did not indicate a statistically significant difference, probably because of the low number of cycles in group A. Furthermore, in group A, the baseline LH:FSH ratio was correlated inversely with the number of oocytes obtained (y = *, r = 0.5, ; Figure 2, line a). On the other hand, as also noticed for the number of follicles, the number of oocytes retrieved was relatively constant, independent of the LH:FSH ratio (v = *, r = 0.05, P = 0.575; Figure 2, line b). However, a comparison of the curves did not indicate a statistically significant difference, possibly also because of the low number of cycles in group A. Analysing the relationship between the baseline LH:FSH value and the maturity of oocytes retrieved, an inverse correlation was noticed in group A between the LHtFSH ratio and the percentage of mature oocytes (y = *, r = 0.845, ; Figure 3, line a). A similar effect was also found in group B, where the LH:FSH ratio was correlated inversely with the percentage of mature oocytes (y = e- 2 *, r = 0.32, P < 0.001; Figure 3, line b). However, a comparison of the slopes of the curves indicated a significantly better correlation between the baseline LH:FSH value and the percentage of mature oocytes in group A than in group B (P < 0.005). Moreover, in patients from group A with a baseline LH:FSH ratio <3, significantly higher numbers of follicles () and oocytes () and percentages of mature oocytes (f < 0.01) were observed (Table I) compared with those with a LH:FSH ratio 3=3, confirming the findings of the linear regression analysis. On the other hand, in group B no difference was observed in the numbers of follicles and oocytes between these two subgroups (Table I), in concert with the results of the linear regression analysis. However, the percentage of mature oocytes decreased significantly () with LH:FSH ratios 2=3 compared with those with ratios <3. Furthermore, the numbers of follicles and oocytes, as well as the percentage of mature oocytes, were similar in patients with a LH:FSH ratio <3 in patients stimulated with HMG and in those stimulated with buserelin-long/hmg (Table I). Conversely, the numbers of follicles and oocytes, as well as oocyte maturity, were significantly higher () in patients with a LH:FSH ratio 5*3 stimulated with buserelinlong/hmg compared with those stimulated with HMG alone, indicating a beneficial effect of the GnRHa administration. In women who miscarried (subgroup Bl, 20/34 women), the mean baseline LH:FSH ratio was significantly higher than in women from subgroup B2 (14/34) who had a live birth (3.3 ± 1.4 versus 1.9 ± 0.6 respectively; P < 0.01). Discussion An impaired baseline LH:FSH ratio represents one of the main endocrinological disturbances in patients with PCOS, resulting from the increased concentrations of LH in this patient population. Raised concentrations of LH are observed in as many as 44% of the patients proven to have PCOS using high-resolution ultrasound (Balen et ai, 1993), although in some women FSH concentrations are also raised (Conway et ai, 1989). Measurements of both FSH and LH concentration in this study were carried out routinely in the same laboratory using the same radioimmunoassay kits. This is an important point when the LH:FSH ratio is employed in the diagnosis of PCOS because the use of different assays may affect the hormone concentration values and consequently the interpretation of the results (Balen et al, 1993). On the other hand, the administration of progestins to induce menses, which may decrease the LH but not the FSH concentrations of the subsequent cycle, should not have a significant impact on our findings, because they were used in a very few cases only. Moreover, despite the existing differences in the patient and cycle numbers, the absence of preselection criteria and of other procedural changes allows the comparison of the two groups. The numbers of follicles developed and oocytes retrieved were found to be inversely associated with the baseline LH:FSH ratio in PCOS patients stimulated with HMG only. This may be a stepwise phenomenon, as indicated by the comparison of patients with LH:FSH ratios <3 and 2=3. This finding probably reflects the intrafollicular endocrinological disturbances described in PCOS. Thus, the increased production of andro- 2547

4 B.C.Tarlatzis etal. Table I. Number of follicles, oocytes and percentage of mature gonadotrophin (HMG) alone or buserelin-long/hmg according oocytes in patients with polycystic ovarian syndrome stimulated with human menopausal to the luteinizing hormone (LH):follicle-stimulating hormone (FSH) ratio (<3 and 3*3) Group A (HMG) LH:FSH Group B (Buserelin-long/HMG) LH:FSH < 3 5=3 Significance a versus b <3 S3 Significance c versus d Follicles (n) 13.1 ± 5.1 a 8.4 ± 4.2 b Oocytes (n) 11.1 ± 5.3 a 5.1 ± 3.9 b Mature oocytes (%) 52.7 a 21.4 b P < ± 6.2 C 12.9 ± 5.8 C ± 7.5 d 12.6 ± 7.5 d 38.7 d NS NS NS = not significant. a compared with c, not significant; b compared with d,. gens by the relatively high LH concentrations coupled with the inefficient aromatization to oestrogens caused by the relatively low FSH concentrations in PCOS patients result in local androgen excess and oestrogen deficit within the ovary (Turhan et al., 1993). This is also supported by the finding of Takahashi et al. (1994) that the LH:FSH ratio is positively correlated with androstenedione in PCOS patients. The increased local androgen concentrations in combination with the local oestrogen deficit constitute a very potent atrogenic environment for the follicle, resulting in the cessation of follicular growth (Turhan et al., 1993). Hence, the higher baseline LH:FSH ratio leads to greater impairment of follicular development. On the other hand, the addition of buserelin in the long desensitization protocol seems to effectively reverse the adverse influence of high basal LH concentrations on follicular development, because the numbers of follicles and oocytes did not decrease with higher LH:FSH ratios. This observation is in accordance with the finding of Turhan et al. (1993) that the suppression of the pituitary-ovarian axis with GnRHa improves follicular synchrony in PCOS patients stimulated for IVF and embryo transfer. Treatment with GnRHa suppresses endogenous LH secretion, preventing follicle exposure to high endogenous gonadotrophins, thus leading to a self-control mechanism in follicular growth and selection without inducing generalized follicular atresia (Lanzone et al., 1987). An improvement in the number of ova after GnRHa treatment in patients with PCOS was also observed by Owen et al. (1989) and Schmutzler et al. (1988), especially in hyperandrogenic patients. This is probably supporting the deleterious effect of androgens on follicular and oocyte development, whereas GnRHa may act either indirectly by suppressing LH, thereby decreasing the LH:FSH ratio and androgen secretion, or directly on the ovary by inhibiting androgen production (Schmutzler et al., 1988). On the other hand, Dor et al. (1992) did not observe a difference in oocyte number between PCOS patients stimulated with FSH/HMG or GnRHa plus FSH/HMG. This could be because of the use of FSH in addition to HMG which may partly correct the LH:FSH ratio in those patients stimulated without GnRHa. The influence of the increased LH concentrations on oocyte quality is an issue of great importance. PCOS patients stimulated for IVF and embryo transfer were found to have a higher number of immature oocytes compared with normo-ovulatory 2548 women (Dor et al., 1992). Our data indicate that oocyte maturity was inversely correlated with the LH:FSH ratio in both stimulation protocols and it was significantly impaired with LH:FSH ratios 3=3. High LH concentrations may interfere with oocyte maturity either directly through its action on the oocyte maturation inhibitor (Balen et al., 1993), or indirectly through androgens (Balen et al., 1993; Watson et al., 1993). The augmented androgen production by LH (Watson et al., 1993; Takahashi et al., 1994) may either lead to follicular atresia, thus decreasing the number of eggs obtained, or adversely affect oocyte quality (Balen et al., 1993; Watson et al., 1993). The administration of GnRHa seems to have a beneficial effect on oocyte quality, as indicated by the comparison of curves between the two groups and of the higher percentage of mature oocytes in patients with a LH:FSH ratio 5=3 in group B than in group A. Nevertheless, GnRHa does not completely correct the adverse effect of impaired ovarian function in PCOS patients, probably because of the incomplete suppression of bioactive LH (Hofmann et al., 1993). This is also supported by the findings of Turhan et al. (1993), who observed an increased number of mature oocytes in PCOS women stimulated with GnRHa for IVF and embryo transfer compared with non-gnrha-stimulated patients. Moreover, Homburg et al. (1993a,) reported better fertilization rates in PCOS patients receiving GnRHa/HMG than in those stimulated with HMG only, which may be further evidence of the better quality of the oocytes obtained. A common troublesome and frustrating complication in PCOS women is the high incidence of early abortions (Balen et al., 1993; Homburg et al., 1993b; Tarlatzis and Grimbizis, 1993). Data from our study show that PCOS patients who miscarried after GnRHa/HMG stimulation had significantly higher LH:FSH ratios than those who had a live birth. A similar comparison was not possible for women stimulated with HMG only because of the low number of pregnancies. It seems, therefore, that the inappropriately raised LH concentration rather than the presence of polycystic ovaries may be the critical factor in determining the risk for miscarriage, as also pointed out by Watson et al. (1993). The use of GnRHa did not prevent this complication probably because of the incomplete suppression of bioactive LH (Hofmann etal., 1993), which exerts its deleterious effect on pregnancy outcome, or because of a longer term effect of high LH concentration

5 exerted on the oocytes prior to GnRHa administration. Thus, pregnancy loss is possibly the consequence of poorer quality embryos resulting from the impaired quality of the oocytes obtained (Balen et al., 1993; Homburg et al., 1993a,b; Watson et al., 1993), as also indicated by the inverse correlation between oocyte maturity and LH:FSH ratio, even in patients receiving GnRHa, as observed in this study. However, it is possible that the increased LH concentration may have an additional effect on the endometrium, inducing abnormal synchronization between decidual maturation, embryonic development and corpus luteum function (Tarlatzis and Grimbizis, 1993; Watson et al., 1993). Nevertheless, Li et al. (1993) failed to detect a correlation between high LH concentration in the follicular phase and endometrial development, suggesting that the association between high LH concentration and poor reproductive performance cannot be explained by abnormal implantation caused by retarded endometrial development. In conclusion, in patients with PCOS infertility stimulated with HMG for ART, a high basal LH:FSH ratio appears to have an adverse effect on follicle and oocyte numbers and quality. On the other hand, the addition of buserelin in the long desensitization protocol seems to reverse the detrimental effect of the increased basal LH concentrations on follicular and oocyte development, because the number of follicles and oocytes did not decrease with higher LH:FSH ratios, whereas the impairment of oocyte maturity is less profound. Additionally, a higher LH:FSH ratio seems to predict a greater possibility for miscarriage, despite the use of GnRHa. 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