Human Reproduction Vol.18, No.11 pp. 2257±2263, 2003
DOI: 10.1093/humrep/deg467
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.Chaf®n1,3, 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
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: [email protected]
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 Bell¯ower Boulevard, Long
Beach, CA 90840, USA
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
Controlled ovarian stimulation (COS) protocols are utilized
extensively to develop multiple pre-ovulatory follicles in the
primate ovary for scienti®c investigation and for collection of
mature oocytes that can be applied to assisted reproductive
technologies (ART) (Cha et al., 2000; Chaf®n et al., 2000). 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., 1984;
Whitman et al., 1989; Goldman et al., 1993; Chaf®n and
Stouffer, 2000). 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 dif®cult (Hartman, 1933;
Corner, 1945; Claman et al., 1993). 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, 1933). In experimental protocols, mistimed aspirations due to variability in follicular phase development can
result in tissue/oocyte collection from an immature or postovulatory 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., 2001);
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
Human Reproduction 18(11) ã European Society of Human Reproduction and Embryology 2003; all rights reserved
2257
K.A.Young et al.
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
n*
FSH: LH
ratioa
hCG
bolusb
Spontaneous
LH surgec
Peak LH
(ng/ml)d
Ovulatory
stigmatae
Progesterone
(ng/ml)f
4
1:1
±
4/4
489 6 160
4/4
4.8 6 1.5
6
1:1
101 6 4
8
2:1
90 6 4
7
1:0
±
+
±
+
±
+
0/3
0/3
0/4
0/4
0/4
0/3
0/3
3/3
0/4
4/4
0/4
0/3
2.0
3.9
0.8
1.9
0.4
2.1
Estradiol level
(pg/ml)
>120
184 6 44
<120
98 6 4
61
68
32
40
9
7
6
6
6
6
6
6
31
34
17
21
2
2
6
6
6
6
6
6
1.6g
2.2
0.6
0.3
0.3
0.6
*Total
number of protocols per treatment.
ratios of FSH:LH were achieved by administering: GnRH antagonist (Antide; 3 mg/kg) plus either:
r-hFSH and r-hLH (30 IU each) (1:1); r-hFSH (30 IU) (1:0); or r-hFSH (30 IU) and r-hLH (15 IU) (2:1).
bFemales were divided to receive either no ovulatory bolus (±) or an ovulatory bolus (+) of hCG (1000 IU rhCG).
cNumber of animals that displayed an LH surge (>150 ng/ml) during the controlled ovulation protocol.
dMean (6SEM) concentrations of LH at time of surge or, in its absence, during FSH:LH treatment before the
hCG bolus.
eNumber of animals displaying an ovulatory stigmata.
fPeak (mean 6 SEM) progesterone level during the subsequent luteal phase of the cycle.
gSee text for explanation of variability.
aVarying
4 and day 8 of the follicular phase; see Goodman et al., 1977;
Pache et al., 1990). 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 re¯ect the steroidogenic development
of the dominant follicle (Bar-Ami, 1994), and ultimately serve
to initiate the pre-ovulatory LH surge (Fink, 1988). 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 (speci®cally LH)
during the ®nal 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, 2000; Levy et al., 2000; Filicori, 2003). Whereas
some studies have concluded that an FSH:LH ratio of 1:0 is
suf®cient to produce viable oocytes (Zelinski-Wooten et al.,
1995; Gordon et al., 2001), others have proposed that inclusion
of LH optimizes ®nal follicular development (e.g. The
European Recombinant Human LH Study Group, 1998;
Gordon et al., 2001; Filicori et al., 2003). 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, 1984). To examine the
role of LH during the ®nal stages of follicular maturation in the
2258
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
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., 1990). 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., 2000).
Hormone assays
Serum concentrations of estradiol and progesterone were determined
by the Endocrine Services Laboratory, ONPRC using speci®c
electrochemoluminescent assays (Roche Elecsys 2010 assay instrument). LH concentrations were determined using a mouse Leydig cell
bioassay (Pau et al., 1993). These assays were validated against
previous radioimmunoassays in the present authors' laboratory (Hess
et al., 1981; Zelinski-Wooten et al., 1991). 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., 1996). 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
Controlled ovulation of the dominant follicle
LH surge in Old World monkeys (Weick et al., 1973). 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 ®nal
gonadotrophin treatment, as reported previously (Hibbert et al.,
1996). 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, 1973). 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.
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 ®mbria (*) 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.
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 ®nal
injection of gonadotrophin alone was administered at 16:00. For the
®nal 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
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 ®nal 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 ®nal 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 signi®cance
level set at 0.05 using the SigmaStat software package (SPSS,
Chicago, IL, USA). To isolate signi®cant differences between groups,
the Student±Newman±Keuls method was used for the pairwise
multiple comparisons.
Results
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 6 44 pg/ml; Table I), treatment failed to prevent a
spontaneous LH surge (4/4 females; 489 6 160 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 preovulatory 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 6 1.5 ng/ml was typical of that observed in the luteal phase
of untreated animals in the colony (Zelinski-Wooten et al.,
1991).
In contrast, when treatment was initiated at estradiol levels
between 80 and 120 pg/ml (mean 98 6 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 6 21
ng/ml; see Table I for individual group details). Although pre2259
K.A.Young et al.
ovulatory follicles typically developed in this group (5/6
females) and estradiol levels remained elevated (211 6 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 6 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 6 2.2 ng/ml in the luteal phase, typical of post-hCG or
post-ovulation progesterone levels (Table I). There was no
signi®cant 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 6 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 6 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 signi®cantly 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 6 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 6 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 6 4 pg/ml;
Table I), the spontaneous LH surge was prevented in all seven
females (mean peak LH value 8 6 2 ng/ml; see Table I for
individual details). The lack of LH administration in this group
resulted in serum LH levels that were signi®cantly 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 signi®cantly lower estradiol levels (49 6 20 pg/ml) as
compared with females in the 1:1 FSH:LH group (211 6 41 pg/
ml) (P < 0.05). Females treated with GnRH antagonist and FSH
alone failed to develop a large antral follicle comparable with
2260
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 6 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
signi®cantly 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 6 123 versus 41 6 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 6 0.6 ng/ml; Table I) to values that
were not signi®cantly different from those of other groups
administered hCG (P > 0.05).
Discussion
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 ®rst 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 ®nal ovulatory maturation of the
dominant follicle.
Rising serum estradiol levels re¯ect the maturity of developing follicles, and trigger the onset of the pre-ovulatory LH
surge (Goodman et al., 1977). 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., 1991).
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., 1991).
Other clinical data have provided evidence that a single dose
(0.5±1.0 mg) of a GnRH antagonist (cetrorelix) prevents a
Controlled ovulation of the dominant follicle
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., 1999). 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 latergeneration GnRH antagonists are more effective in preventing
the spontaneous LH surge at any time during the follicular
phase, whereas smaller doses may have more ef®cacy 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 de®ned, initiating one treatment protocol at an
estradiol level of 70 pg/ml (data not shown) resulted in a
grossly atretic follicle with a blood-®lled 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.,
1991); 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 6 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, 1989; Filicori et al., 2002).
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, 1998). 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 signi®cantly 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
®nal follicle development, proper steroidogenesis and normal
luteinization in both human and non-human primate studies
(Seibel et al., 1982; Zeleznik and Hillier, 1984; ZelinskiWooten et al., 1991; Weston et al., 1996; Filicori et al., 2003).
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 preovulatory antral follicle development and ovulation were
prevented in the FSH-only females, suggesting that LH action
is necessary in rhesus monkeys during the ®nal 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 ®nal follicle
maturation (e.g. Sullivan et al., 1999; Filicori and Cognigni
2001), and that the addition of LH to FSH-only protocols in the
mid to late follicular phase can enhance follicle growth
(Filicori et al., 1999). The present data indicate for the ®rst 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 (Chaf®n
and Stouffer, 1999; Duffy and Stouffer, 2001; Stouffer et al.,
2001). In addition, this model is ideal for future studies
examining the requirement of LH in ®nal follicular maturation.
This model, in controlling the dominant follicle, complements
existing COS protocols (e.g. Chaf®n and Stouffer, 1999; Cha
et al., 2000; Chaf®n et al., 2000), and will allow systematic
comparisons between oocytes and other tissues/cells taken
from the dominant follicle of controlled ovulation cycles, and
2261
K.A.Young et al.
those from multiple pre-ovulatory follicles of COS cycles in
non-human primates.
In conclusion, the ®nal 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.
Speci®cally, 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 ®nal
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-HD07133) and NICHD HD042896 (K.A.Y.).
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Submitted on May 13, 2003; resubmitted on July 7, 2003; accepted on
August 6, 2003
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