Controlled ovulation of the dominant follicle: a critical role for LH in the late follicular phase of the menstrual cycle

Kelly A. Young1,,4, Charles L. Chaffin1,,3, Theodore A. Molskness1 and Richard L. Stouffer1,,2

1 Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, USA 3 Current address: Medical College of Georgia, Department of Physiology, CL2126, 1120 15th St Augusta, GA 30921, USA 4 Current address: California State University, Long Beach, Department of Biological Sciences, 125 Bellflower Boulevard, Long Beach, CA 90840, USA

2 To whom correspondence should be addressed at: Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 NW 185th Ave, Beaverton, Oregon 97006, USA. e-mail: stouffri{at}ohsu.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: A method was sought to control ovulation of the dominant follicle and to test the importance of LH during the late follicular phase of the menstrual cycle. Menstrual cycles of rhesus monkeys were monitored, and treatment initiated at the late follicular phase (after dominant follicle selection, before ovulation). METHODS: The 2-day treatment consisted of GnRH antagonist plus either r-hFSH and r-hLH (1:1 or 2:1 dose ratio) or r-hFSH alone. In addition, half of the females received an ovulatory bolus of hCG. RESULTS: When treatment was initiated at estradiol levels >120 pg/ml, neither the endogenous LH surge, ovulation nor luteal function were controlled. However, when treatment was initiated at estradiol levels 80–120 pg/ml using either 1:1 or 2:1 dose ratios of FSH:LH, the LH surge was prevented, and ovulation occurred following hCG treatment. FSH-only treatment also prevented the LH surge, but follicle development appeared abnormal, and hCG failed to stimulate ovulation. CONCLUSIONS: Control over the naturally dominant follicle is possible during the late follicular phase using an abbreviated GnRH antagonist, FSH+LH protocol. This method offers a model to investigate periovulatory events and their regulation by gonadotrophins/local factors during the natural menstrual cycle in primates.

Key words: dominant follicle/GnRH antagonist/gonadotrophins/LH/ovarian stimulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Controlled ovarian stimulation (COS) protocols are utilized extensively to develop multiple pre-ovulatory follicles in the primate ovary for scientific investigation and for collection of mature oocytes that can be applied to assisted reproductive technologies (ART) (Cha et al., 2000Go; Chaffin et al., 2000Go). The administration of supraphysiological concentrations of exogenous gonadotrophins in COS cycles overrides the natural process of dominant follicle selection and stimulates the development of numerous large antral follicles. However, these follicles are heterogeneous in terms of quality and response to ovulatory stimulation, and vary in both oocyte maturity and somatic (granulosa) cell differentiation (Laufer et al., 1984Go; Whitman et al., 1989Go; Goldman et al., 1993Go; Chaffin and Stouffer, 2000Go). Ideally, investigations on the cellular and molecular events during ovulation and luteinization, and perhaps selected ART procedures on the mature oocyte, would utilize the biologically dominant follicle of the normal menstrual cycle. However, natural variation in the interval for follicle maturation (e.g. the length of the follicular phase) and the timing of the pre-ovulatory LH surge among non-human primates and women makes follicle sampling during the spontaneous cycle logistically difficult (Hartman, 1933Go; Corner, 1945Go; Claman et al., 1993Go). While the modal length of the follicular phase in rhesus monkeys is 12–13 days, the pre-ovulatory phase can range between 9 and 17 days (Hartman, 1933Go). In experimental protocols, mistimed aspirations due to variability in follicular phase development can result in tissue/oocyte collection from an immature or post-ovulatory follicle.

In the present study, a non-human primate model was sought that would control ovulatory timing and hence permit future experimental analysis of the dominant follicle at precise stages of the periovulatory interval in the menstrual cycle. Serial ultrasound scans have been utilized in women to determine the stage of follicle development (e.g. Vlaisavljevic et al., 2001Go); however, herein a model was sought where ovulatory control could be established with precise administration of a GnRH antagonist and gonadotrophins. In order to closely mimic natural conditions, an acute (2-day) protocol of GnRH antagonist plus gonadotrophin replacement was chosen which began after selection of the dominant follicle (i.e. between day 4 and day 8 of the follicular phase; see Goodman et al., 1977Go; Pache et al., 1990Go). The dominant follicle was considered to be controlled when: (i) a single, large antral follicle developed; (ii) the natural LH surge was prevented; and hence (iii) ovulation and luteinization did not occur unless an hCG bolus was administered.

In order to overcome the diversity in follicular phase lengths between both individuals and menstrual cycles, it was predicted that serum estradiol levels could be used to standardize the onset of controlled ovulation protocols. Rising estradiol levels reflect the steroidogenic development of the dominant follicle (Bar-Ami, 1994Go), and ultimately serve to initiate the pre-ovulatory LH surge (Fink, 1988Go). Therefore, the initial step in controlled ovulation model development was to ascertain the optimal serum estradiol levels at which to begin GnRH anatagonist and gonadotrophin replacement. After optimal estradiol concentrations for controlled ovulation treatment were determined, further studies were performed to analyse the requirements for gonadotrophins (specifically LH) during the final stages of follicular maturation prior to ovulation.

The role of LH in controlled follicle maturation has been debated in both human and non-human primate studies (Howles, 2000Go; Levy et al., 2000Go; Filicori, 2003Go). Whereas some studies have concluded that an FSH:LH ratio of 1:0 is sufficient to produce viable oocytes (Zelinski-Wooten et al., 1995Go; Gordon et al., 2001Go), others have proposed that inclusion of LH optimizes final follicular development (e.g. The European Recombinant Human LH Study Group, 1998Go; Gordon et al., 2001Go; Filicori et al., 2003Go). Indeed, FSH stimulates LH receptor expression in granulosa cells of developing follicles, suggesting a role for LH in the late follicular phase (Zeleznik and Hillier, 1984Go). To examine the role of LH during the final stages of follicular maturation in the present 2-day controlled ovulation protocol, the amount of FSH was held constant while the LH dose was varied to produce FSH:LH ratios of 1:1, 2:1 and 1:0 prior to hCG bolus administration.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
All protocols were approved by the Oregon National Primate Research Center (ONPRC) Animal Care and Use Committee, and conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. A description of the care and housing of rhesus monkeys (Macaca mulatta) at the ONPRC was published previously (Wolf et al., 1990Go). Menstrual cycles of adult female rhesus monkeys (n = 25) were monitored daily, and blood samples were collected by saphenous venipuncture daily starting 6 days after the onset of menses until the next menstrual period (Duffy et al., 2000Go).

Hormone assays
Serum concentrations of estradiol and progesterone were determined by the Endocrine Services Laboratory, ONPRC using specific electrochemoluminescent assays (Roche Elecsys 2010 assay instrument). LH concentrations were determined using a mouse Leydig cell bioassay (Pau et al., 1993Go). These assays were validated against previous radioimmunoassays in the present authors’ laboratory (Hess et al., 1981Go; Zelinski-Wooten et al., 1991Go). Previous experiments from this laboratory have shown the pre-ovulatory peak of bioactive LH to fall within the 300–600 ng/ml range, with serum LH values generally being maintained between 15 and 35 ng/ml for the remainder of the cycle (Molskness et al., 1996Go). Based on a conservative estimate, LH levels >150 ng/ml were considered indicative of an LH surge.

Study 1: Controlled ovulation protocol
To determine when to begin treatment, the initial protocol was started at a variety of estradiol levels during the mid-to-late follicular phase of natural cycles (days 6–13 of cycle, average day 10; n = 10 cycles for initial protocol). The initial protocol consisted of four sets of injections of a GnRH antagonist and a FSH:LH 1:1 dose ratio over 2 days. On day 1 of treatment, GnRH antagonist (Antide; Ares Serono Group, Ltd; 3 mg/kg), r-hFSH and r-hLH (30 IU each; Ares Serono Group, Ltd) were administered at 08:00; gonadotrophins alone were administered at 16:00. On day 2, GnRH antagonist (Antide; 0.5 mg/kg) and gonadotrophins were administered at 08:00, and a final injection of gonadotrophin alone was administered at 16:00. For the final injection, females were divided arbitrarily to receive either no ovulatory bolus (FSH + LH at 16:00) or an ovulatory bolus of hCG (1000 IU r-hCG at 16:00). Ovulation generally occurs by 36 h after the LH surge in Old World monkeys (Weick et al., 1973Go). In order to prevent potentially missing the ovulation event, the ovaries were viewed via laparoscopy for the presence of follicles and evidence of follicle rupture (protruding stigmata) at 72 h after the final gonadotrophin treatment, as reported previously (Hibbert et al., 1996Go). Laparoscopic examination of ovaries is a useful method to determine the extent of late follicle development and to discern if ovulation has occurred (Rawson and Dukelow, 1973Go). Ovaries with structures (ovulatory stigmata, developing follicles) were compared with contralateral ovaries for size and degree of vasculature differences; surgeries were recorded digitally to compare results in a single analysis.

Study 2: Analysis of FSH:LH ratios
Following the establishment of optimal estradiol levels for initiating the treatment protocol, additional animals were recruited to consider the effects of different FSH:LH ratios in the controlled ovulation model. To determine the role of LH in the final pre-ovulatory follicle maturation, females were treated with GnRH antagonist and gonadotrophins, as described above, but the FSH:LH ratio was altered. FSH:LH ratios common in clinical protocols were utilized. One group (n = 8 cycles) received Antide, FSH, plus half the amount of LH given previously; 30 IU FSH:15 IU LH (FSH:LH 2:1); a second group (n = 7 cycles) received Antide plus 30 IU FSH alone (FSH:LH 1:0). Finally, the response to hCG was monitored in females administered FSH:LH ratios of 2:1 and 1:0. These females were treated as above, with the final injection of gonadotrophins replaced by an ovulatory bolus of hCG.

Statistical analysis
Statistical evaluation of mean differences among experimental groups was performed by ANOVA or ANOVA on Ranks with a significance level set at 0.05 using the SigmaStat software package (SPSS, Chicago, IL, USA). To isolate significant differences between groups, the Student–Newman–Keuls method was used for the pairwise multiple comparisons.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study 1: Optimal estrogen level for controlled ovulation initiation

When GnRH antagonist and FSH:LH (1:1) treatment was initiated at an estradiol level >120 pg/ml (mean serum level 184 ± 44 pg/ml; Table I), treatment failed to prevent a spontaneous LH surge (4/4 females; 489 ± 160 ng/ml). In all females in this group, follicle rupture was not prevented and occurred spontaneously as demonstrated by ovulatory stigmata viewed at the time of laparoscopy (Figure 1A). Single pre-ovulatory follicles developed in these females, and no antral follicle development was apparent on the contralateral ovary of any animal, despite administration of exogenous gonadotrophins (Figure 1B). Normal luteal phases occurred in all four females in this group, and the mean peak progesterone level of 4.8 ± 1.5 ng/ml was typical of that observed in the luteal phase of untreated animals in the colony (Zelinski-Wooten et al., 1991Go).


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Table I. Summary of results from controlled ovulation protocols (with or without an hCG bolus) initiated at circulating serum estradiol levels >120 or <120 pg/ml
 


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Figure 1. Laparoscopic views of rhesus monkey ovaries 72 h after initiation of controlled ovulation protocols described in this study. These photographs depict typical ovarian response following GnRH antagonist treatment at estradiol (E2) levels >120 pg/ml (panels A and B) or <120 pg/ml (panels C–F), plus various FSH:LH ratios without (panels A, C and E) or with (panels D and F) an ovulatory hCG bolus. (A) E2 level >120 pg/ml; GnRH antagonist + FSH:LH (1:1), no hCG bolus. Ovulation site on rhesus monkey ovary (o) stimulated with controlled ovulation protocol starting at E2 > 120 pg/ml. The single, red, raised stigmata (s) is evidence for dominant follicle ovulation. Adjacent oviductal fimbria (*) are also visible. The inset depicts detail of a raised stigmata. (B) Ovary (o) contralateral to one shown in panel (A) with adjacent uterus (u). The contralateral ovary bears no visible large antral follicles or stigmata. (C) E2 level < 120pg/ml; FSH:LH (1:1), no hCG bolus. Ovary with single, developed, pre-ovulatory follicle (f). No ovulatory stigmata was observed in controlled ovulation protocols starting at E2 < 120 pg/ml without administration of an hCG bolus. (D) FSH:LH 2:1; E2 level < 120 pg/ml, +hCG. Administration of hCG to the 2:1 FSH:LH-treated females resulted in ovulation. A post-ovulatory stigmata is depicted extruding from the ovary wall. (E) E2 level < 120 pg/ml; FSH:LH (1:0), no hCG. Both panels depict small follicles that developed in females stimulated without LH. The ovaries in females in this group failed to develop mature pre-ovulatory follicles; no ovulatory stigmata were noted. (F) FSH:LH 1:0; E2 level < 120 pg/ml, + hCG. Administration of hCG to females treated without LH failed to produce normal ovulation. There was no evidence of ovulatory rupture of the large antral follicles found in females in this group; however, degenerated small follicles (d) are apparent at the ovarian surface. The panel depicts ovaries from two females in this treatment group.

 
In contrast, when treatment was initiated at estradiol levels between 80 and 120 pg/ml (mean 98 ± 4 pg/ml; Table I), the spontaneous pre-ovulatory LH surge was prevented during the treatment protocol in all six animals (peak LH value 64 ± 21 ng/ml; see Table I for individual group details). Although pre-ovulatory follicles typically developed in this group (5/6 females) and estradiol levels remained elevated (211 ± 41 pg/ml for group after 1 day of treatment), spontaneous ovulation was prevented in the absence of an ovulatory hCG bolus (3/3 females) (Figure 1C). In two of the three females not administered hCG, serum levels of progesterone remained near baseline with peak value of 0.5 ± 0.4 ng/ml during the luteal phase, and were lower than progesterone levels in females starting treatment protocols at an estradiol level >120 pg/ml (P < 0.05). The remaining female not administered hCG had progesterone concentrations rising to 5.0 ng/ml (see Table I for group values). In females administered hCG, ovulation was observed (3/3 females), and the mean peak progesterone level was 3.9 ± 2.2 ng/ml in the luteal phase, typical of post-hCG or post-ovulation progesterone levels (Table I). There was no significant difference in progesterone levels among females administered the FSH:LH (1:1) protocol (P = 0.6), nor in the length of the luteal phase among females, regardless of the treatment group (P = NS; data not shown).

Study 2: The contribution of LH to the controlled ovulation protocol
FSH:LH 2:1
When GnRH antagonist and FSH+LH (2:1) treatment was initiated at estradiol levels between 80 and 120 pg/ml (101 ± 4 pg/ml; Table I), a single large pre-ovulatory follicle was observed in seven of the eight females, but a spontaneous LH surge was prevented in all eight females in this group (peak LH values 36 ± 13 ng/ml; see Table I for individual details). Serum LH levels in this group did not differ from that of females in either the FSH:LH 1:1 treatment group (P = 0.25) or the FSH:LH (1:0) group (P = 0.055). Serum estradiol levels did not differ significantly from females in the 1:1 or 1:0 treatment groups on either day 1 or 2 of treatment (data not shown; P = NS in all cases). In females not administered an ovulatory bolus of hCG, ovulation and luteinization were prevented (4/4 females; data not shown), and mean peak progesterone levels were low throughout the luteal phase (0.8 ± 0.6 ng/ml). However, the administration of an hCG bolus promoted ovulation in the FSH:LH (2:1)-treated females (Figure 1D). All (4/4) females examined by laparoscopy displayed ovulatory stigmata on one ovary and had progesterone levels that were normal for post-hCG administration (1.9 ± 0.3 ng/ml) (Table I).

FSH:LH 1:0
When GnRH antagonist and FSH alone were administered at estradiol levels between 80 and 120 pg/ml (90 ± 4 pg/ml; Table I), the spontaneous LH surge was prevented in all seven females (mean peak LH value 8 ± 2 ng/ml; see Table I for individual details). The lack of LH administration in this group resulted in serum LH levels that were significantly lower than those noted in the FSH:LH (1:1) females (P < 0.05). After one day of treatment, females treated with the 1:0 FSH:LH protocol had significantly lower estradiol levels (49 ± 20 pg/ml) as compared with females in the 1:1 FSH:LH group (211 ± 41 pg/ml) (P < 0.05). Females treated with GnRH antagonist and FSH alone failed to develop a large antral follicle comparable with that seen in animals administered both FSH and LH (Figure 1E). In FSH:LH 1:0-treated females that did not receive an ovulatory bolus of hCG (n = 4), spontaneous ovulation and luteinization were prevented. The mean progesterone level was low throughout the luteal phase (0.4 ± 0.3 ng/ml) in this group. When an ovulatory bolus of hCG was administered to females in the 1:0 FSH:LH group, follicle rupture was not apparent. No ovulatory stigmata were noted in the ovaries in these females (n = 3; Figure 1F). Notably, the ovaries in this treatment group all displayed evidence of degenerated small antral follicles on the ovarian surface (Figure 1F). Whereas administration of hCG did not significantly increase serum estradiol levels in the 1:1 or 2:1 treatment groups (data not shown), the hCG bolus increased estradiol levels more than 5.6-fold (231 ± 123 versus 41 ± 28 pg/ml, day 1 post-hCG versus no hCG) in the 1:0 FSH:LH group. Progesterone concentrations also rose post-hCG administration in this group (2.1 ± 0.6 ng/ml; Table I) to values that were not significantly different from those of other groups administered hCG (P > 0.05).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovulatory control over the naturally selected dominant follicle (controlled ovulation; COv) appears possible using an acute treatment protocol within a narrow interval during follicular maturation in the late follicular phase of the menstrual cycle in rhesus monkeys. At estradiol levels of 80–120 pg/ml, suppressing GnRH action while administering gonadotrophins maintains the dominant follicle. Acute (48 h) treatment during this interval prevented both the spontaneous LH surge and ovulation, while allowing initiation of periovulatory events, including follicle rupture and luteinization, following administration of an hCG bolus. The present study was the first to establish this window of opportunity, as determined by serum estradiol levels, during the follicular phase to provide ovulatory control of the single dominant follicle in the rhesus macaque. In addition, the results suggest that removal of LH support within this window prevents final ovulatory maturation of the dominant follicle.

Rising serum estradiol levels reflect the maturity of developing follicles, and trigger the onset of the pre-ovulatory LH surge (Goodman et al., 1977Go). In the present study, initiating the GnRH antagonist and FSH:LH (1:1) controlled ovulation protocol at an estradiol level >120 pg/ml typically failed to prevent spontaneous ovulation of the dominant follicle. Essentially, Antide and gonadotrophin treatment was administered too late in these females, and/or the dose of Antide was not enough to prevent the ongoing LH surge. The spontaneous LH surge is not prevented in stump-tailed macaques during the mid-follicular stage (day 10, rising estradiol levels) by the administration of 1 mg/kg Antide (Fraser et al., 1991Go). However, in women the administration (10 mg) of the GnRH antagonist detirelix prevented a spontaneous LH surge during both the mid-follicular and pre-ovulatory (LH surge already initiated) stages of the menstrual cycle (Fluker et al., 1991Go). Other clinical data have provided evidence that a single dose (0.5–1.0 mg) of a GnRH antagonist (cetrorelix) prevents a spontaneous LH surge in most women, provided that the antagonist is administered when plasma estradiol levels are 100–150 pg/ml (Rongieres-Bertrand et al., 1999Go). These data suggest that a dose or drug difference may exist for controlling the LH surge with antagonist, as well as a potential difference among primate species. It appears that the larger doses of later-generation GnRH antagonists are more effective in preventing the spontaneous LH surge at any time during the follicular phase, whereas smaller doses may have more efficacy when estradiol levels have not peaked.

Ovulatory control was achieved when the GnRH antagonist FSH:LH (1:1) controlled ovulation protocol was started at an estradiol level <120 pg/ml, as determined by the prevention of a spontaneous LH surge, and lack of ovulation in the absence of an ovulatory hCG bolus. Utilizing serum estradiol levels to determine follicular maturity circumvents the daily ultrasound or LH bioactivity assays used in stimulation protocols to determine follicle development and ovulatory timing a priori. Although the lower limits of the present estradiol window were not critically defined, initiating one treatment protocol at an estradiol level of 70 pg/ml (data not shown) resulted in a grossly atretic follicle with a blood-filled antrum, suggesting that the estradiol range of 80–120 pg/ml is optimal for this acute protocol. The administration of Antide alone in the late follicular phase prevents ovulation in macaques (Fraser et al., 1991Go); the gonadotrophins administered in the present study maintained the antral follicle and allowed ovulation with an hCG bolus. These exogenous gonadotrophins did not, however, stimulate development of multiple large antral follicles, as a single follicle was noted on only one ovary in all groups examined (see representative contralateral ovary, Figure 1B).

Despite markedly different progesterone levels, there were no differences in luteal phase length among all females that did not undergo oophorectomy after the treatment had been concluded (n = 17); the average time from treatment termination to menstruation in all groups was 12.5 ± 1 days. In the absence of an ovulatory hCG bolus, there were no apparent ovulations or notable luteal phases, save one exception, as judged by the near-baseline serum progesterone. An elevated progesterone level was noted for this female following the protocol, although an ovulatory stigmata was not observed on the large follicle. Elevations in progesterone level typically occur post-ovulation; therefore, this female may have experienced an abbreviated LH surge that triggered a temporary rise in progesterone. Alternatively, the FSH:LH (1:1) protocol may have induced luteinization of the unruptured follicle in this female, as data suggest that high levels of LH or FSH can correlate with progesterone secretion in the late follicular phase (Opavsky and Armstrong, 1989Go; Filicori et al., 2002Go).

Some protocols used to promote follicular development in anovulatory females recommend a 2:1 ratio of FSH:LH for successful stimulation of women lacking natural GnRH activity (e.g. The European Human Recombinant LH Study Group, 1998Go). In the present study, monkeys receiving 2:1 FSH:LH treatment where the amount of LH given was reduced by half, developed single mature pre-ovulatory follicles. In the absence of the hCG bolus, the natural LH surge and ovulation were circumvented with this protocol, and progesterone levels were not elevated during the luteal phase. Importantly, when monkeys were treated with the 2:1 FSH: LH ratio plus a bolus of hCG, controlled ovulation occurred, suggesting that reducing the amount of administered LH did not diminish the ability of the ovary to respond to an ovulatory stimulus. Indeed, progesterone levels in monkeys administered 2:1 FSH:LH + hCG were not significantly different from those seen in females of the 1:1 FSH:LH + hCG group, or in the >120 pg/ml estradiol protocol initiating group where spontaneous ovulation occurred (P > 0.05). It appears that the 2:1 ratio was as effective as the 1:1 ratio for stimulating the dominant follicle in rhesus monkeys during the present controlled ovulation protocol.

The expression of LH receptors and LH action on the antral follicle has been cited as being advantageous for aspects of final follicle development, proper steroidogenesis and normal luteinization in both human and non-human primate studies (Seibel et al., 1982Go; Zeleznik and Hillier, 1984Go; Zelinski-Wooten et al., 1991Go; Weston et al., 1996Go; Filicori et al., 2003Go). In order to examine the role of LH in follicle maturation during controlled ovulation protocols, data from the FSH:LH 1:1 and 2:1 ratio groups were compared to that from a group receiving FSH only (1:0 FSH:LH). A spontaneous LH surge, normal pre-ovulatory antral follicle development and ovulation were prevented in the FSH-only females, suggesting that LH action is necessary in rhesus monkeys during the final stages of antral follicle development (Figure 1E and F). Indeed, the females in the 1:0 FSH:LH group were unique in their non-responsiveness to a standard ovulatory stimulus, as hCG administration in the present study failed to elicit follicle rupture. Interestingly, estradiol and progesterone levels were increased among 1:0 FSH:LH females administered hCG compared with cohorts not exposed to an ovulatory bolus. This suggested that, whilst these follicles failed to ovulate, they were responsive to LH. The present data are consistent with other reports suggesting that LH action on antral follicles is necessary for final follicle maturation (e.g. Sullivan et al., 1999Go; Filicori and Cognigni 2001Go), and that the addition of LH to FSH-only protocols in the mid to late follicular phase can enhance follicle growth (Filicori et al., 1999Go). The present data indicate for the first time the requirement for LH to stimulate antral follicle maturation in the late stages of the follicular phase of the natural menstrual cycle.

A model for controlled ovulation during the spontaneous menstrual cycle provides a novel and important method to examine periovulatory events in the naturally selected dominant follicle in a time-dependent manner. This model can be used in non-human primates to further elucidate the factors regulating the primate periovulatory follicle, including steroids, prostaglandins, proteases and angiogenic factors (Chaffin and Stouffer, 1999Go; Duffy and Stouffer, 2001Go; Stouffer et al., 2001Go). In addition, this model is ideal for future studies examining the requirement of LH in final follicular maturation. This model, in controlling the dominant follicle, complements existing COS protocols (e.g. Chaffin and Stouffer, 1999Go; Cha et al., 2000Go; Chaffin et al., 2000Go), and will allow systematic comparisons between oocytes and other tissues/cells taken from the dominant follicle of controlled ovulation cycles, and those from multiple pre-ovulatory follicles of COS cycles in non-human primates.

In conclusion, the final maturation and ovulation of the naturally selected dominant follicle in rhesus monkeys can be controlled when GnRH antagonist and gonadotrophin replacement protocol is initiated at an estradiol level of 80–120 pg/ml. Specifically, the 30 IU FSH:15 IU LH (2:1 FSH:LH) protocol allows induction of ovulation with an hCG bolus, while not promoting luteinization of the unruptured follicle. Importantly, the present data also suggest an essential role for LH in final follicular maturation in the natural cycle. This method offers a potential model to facilitate investigation of temporal events in the dominant follicle, without the use of daily ultrasound/anaesthesia administration, and their regulation by gonadotrophins or local factors during the periovulatory interval in the menstrual cycle.


    Acknowledgements
 
The authors are grateful for the expert contributions of the Division of Animal Resources, the Endocrine Services Core, the Molecular and Cellular Biology Core Laboratory, and the ONPRC surgery team. Gonadotrophins were generously donated by the Ares Serono group. This research was supported by NIH NICHD HD20869 (R.L.S.), through a cooperative agreement (U54-HD18185) as part of the Specialized Cooperative Centers Program in Reproductive Research, NCRR RR00163 (R.L.S.), NIH NICHD Training Grant (T32-HD- 07133) and NICHD HD042896 (K.A.Y.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 13, 2003; resubmitted on July 7, 2003; accepted on August 6, 2003.