Download Evidence of `genuine empty follicles` in follicular

Hum. Reprod. Advance Access published January 27, 2009
Human Reproduction, Vol.1, No.1 pp. 1– 5, 2009
doi:10.1093/humrep/den497
CASE REPORT Reproductive endocrinology
Evidence of ‘genuine empty follicles’ in
follicular aspirate: a case report†
N. Desai 1, C. Austin, F. AbdelHafez, J. Goldfarb, and T. Falcone
Cleveland Clinic Foundation, Department of OB/GYN, 26900 Cedar Road, Beachwood, OH, 44122, USA
1
Correspondence address. Tel: þ216-839-3150; Fax: þ216 839-3180; E-mail: [email protected]
Failure to retrieve oocytes after normal ovarian stimulation has been labeled ‘empty follicle syndrome’ (EFS). The existence of genuine EFS
has been questioned and is still controversial. Here, we report an unusual case in which an extraordinary number of empty follicle-like structures were identified in the ovarian aspirate at the time of retrieval. A 31-year-old woman presented with a 4-year history of primary infertility and underwent ovulation induction. The patient was given hCG and oocyte retrieval was performed 36 h later. During the oocyte
retrieval, more than 200 tiny structures resembling pre-antral follicles were noted in the ovarian aspirate. They exhibited two to three
layers of granulosa cells and appeared to enclose an immature oocyte. They showed a great variation in size ranging between 40 and
80 mm. These structures were further characterized by electron microscopy and cultivated in vitro to assess hormone secretion. The follicles
were found to be devoid of oocytes, but each had a readily identifiable zona. Hormone assays revealed that these follicles were secreting
increasing levels of estradiol. A second in vitro fertilization attempt gave similar results. These data are suggestive of some failure in the oocyte
maturation process. We speculate that this may be the first actual evidence to support the existence of true empty follicles, which if left to
grow in vivo might lead to empty graffian follicles and genuine EFS.
Key words: empty follicle syndrome / ovulatory dysfunction / in vitro fertilization / zona pellucida
Introduction
Failure to retrieve oocytes after normal ovarian stimulation has been
labeled ‘empty follicle syndrome’ (EFS). It was described for the first
time by Coulam et al. (1986). Incidence of EFS ranges from ,1
(Awonuga et al., 1998) to 7% (Bartfai et al., 1987). This syndrome is
not well characterized and its cause remains controversial. Although
rare, failure of oocyte retrieval is very stressful for both the patient
and the physician. Three main causes have been suggested for the pathogenesis of this syndrome: ovarian dysfunction, iatrogenic causes and
technical causes. Iatrogenic causes may be due to the use of inappropriate batches, dosing or timing of hCG which is injected for the final steps
of maturation (reviewed in Bustillo (2004)). Some investigators question
the existence of ‘genuine EFS’ and prefer to call it unsuccessful oocyte
retrieval as they believe that iatrogenic and technical causes are responsible in almost all cases of EFS (Aktas et al., 2005). The term ‘borderline
form of EFS’ has been applied to describe cases where very few mature
or immature oocytes are retrieved from the aspiration of several follicles
after a satisfactory ovarian stimulation regimen (Isik and Vicdan, 2000;
Nikolettos et al., 2004).
Here, we report a case of a patient undergoing routine in vitro fertilization (IVF) retrieval who had an extraordinary number of empty folliclelike structures in her follicular aspirate. In a subsequent IVF cycle, these
empty follicles were once again observed. This may be the first documentation of ‘true empty follicles’ at their earliest developmental stage.
Case report
The patient is a nulligravida woman who presented at an age of 29
with a 4-year history of infertility. Her menstrual cycles were
regular: 26 –28 days in length. She described her periods as 7 days
long, heavy, with moderate dysmenorrhea. She was on no medications
and had no history of prior abdominal surgery. Hysterosalpingogram
(HSG) revealed bilateral fill and spill of the fallopian tubes, but the
fundus had a concave shape suggestive of an extrinsic pressure
effect. Ultrasound confirmed multiple uterine fibroids including a
large posterior one measuring 51 46 mm.
The patient was treated empirically with two cycles of clomid with
intrauterine insemination before undergoing an abdominal myomectomy. Following her surgery, the couple attempted to conceive
without treatment for approximately six cycles. When she returned
to resume treatment, a repeat HSG was performed revealing a
normal uterine cavity, fill and spill of the left fallopian tube, but only
partial filling of the right tube. She completed one more cycle of
clomid with insemination.
†Presented in part at the 64th annual meeting of the American Society for Reproductive Medicine, 8–12 November 2008, San Francisco, CA, USA.
& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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Desai et al.
The couple then decided to change their treatment to IVF. The
patient was 31 at the time of her first IVF attempt. Ovarian stimulation
was carried out using the long luteal phase Lupron protocol with
200 U of recombinant FSH for 10 days. The peak estradiol level on
Day 11 was 1779 pg/ml. Seven follicles were documented by ultrasound, measuring 17–20.5 mm average diameter. The patient was
given hCG (Ovidrel) on the evening of Day 11, and oocyte retrieval
was scheduled 36 h later. Transvaginal ultrasound-guided follicular
aspiration was performed using a standard double lumen needle.
The process of oocyte retrieval was uneventful and 14 oocytes
were recovered. Only three oocytes were mature and two were fertilized after intracytoplasmic sperm injection. The embryos were
transferred to the patient’s uterus on Day 3 at the 7–9-cell stage.
What was striking about this case was that during the oocyte
retrieval, the embryologist noted 20–30 tiny follicle-like structures
in each tube of follicular aspirate. These structures were examined
at 300 magnification using an inverted microscope (Fig. 1). The
follicles showed a great variation in size, ranging from 40 to 80 mm
in diameter. They appeared to have an intact thecal layer and a
zona. We did notice that the central region with the ‘oocyte’ appeared
a little odd: it was smooth with a clear appearance. The pooled
aspirates contained over 200 of these pre-antral follicles.
Convinced that these were indeed primary and secondary follicles,
we placed some follicles in in-vitro maturation medium (IVM) medium,
vitrified some for future studies and fixed some for transmission electron microscopy. The IVM medium was minimum essential medium
with 1% protein supplemented with insulin (10 mg/ml) and FSH
(100.5 mIU/ml). The follicles attached to the culture surface within
24 h of plating. Granulosa cell (GC) proliferation was demonstrated
by an increase in diameter of plated follicles. An hormone profile
revealed increasing levels of estradiol secretion starting from 152
(8 pg/follicle plated) to 1462 pg/ml (or 77 pg per follicle) after 13
days of culture. Progesterone was not detected in the follicle culture
supernatants. We examined few follicles after just 24 h of in vitro
culture. Follicles were treated with hyaluronidase to remove
cumulus cells and to determine nuclear status of the oocyte. Interestingly, all of the follicles examined were determined to be devoid of an
oocyte; yet there was clear evidence of a very thin zona pellucida
(Fig. 2). This was later corroborated by the electron microscopy
studies (Fig. 3).
The patient did not become pregnant and underwent a second IVF
cycle. In light of the poor yield of mature oocytes and the bizarre,
unexplained presence of so many pre-antral -like follicles in her first
cycle, a different IVF stimulation regimen was attempted. Luteinizing
hormone (LH) was added to the stimulation protocol, the follicles
were allowed to grow to a larger diameter before administration of
hCG and the retrieval was scheduled for 38 h after hCG rather than
36 h, which is our standard practice. The patient was down-regulated
with Lupron and stimulated with 225 U of recombinant FSH and 75 U
of recombinant LH for 11 days.
Her peak estradiol level was 1794 pg/ml on Day 11. On an ultrasound scan, seven follicles were seen and measured 19–22 mm in
diameter. We retrieved nine oocytes; six were mature and injected
with husband’s sperm. Four of these oocytes were fertilized. Two
embryos were transferred at the seven and five cell stage and none
were frozen. Again, the patient did not become pregnant.
Figure 1 Photomicrographs of follicle-like structures observed at the time of oocyte retrieval. Over 200 or more structures were recovered from
the ovarian aspirates. A wide range of sizes were observed; they ranged from 40 to 80 mm in diameter.
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Evidence of empty follicles
Figure 2 The follicle-like structures after treatment with hyaluronidase to remove GCs. Structures were devoid of oocytes but thin zonae were
clearly visualized.
Figure 3 Transmission electron micrographs of follicle-like structures. (A) Follicle-like structure composed of GCs but no oocyte (800). (B) Inset
magnified showing cytoplasmic organelles within a GC; MV: microvilli; ER: endoplasmic reticulum; M: mitochondria (22 000).
Once again follicle-like structures were observed in the follicular
aspirate, although in far lower numbers than in the first cycle,
possibly due to the changes in the stimulation protocol. This time
the ‘follicle-like’ structures were treated with hyaluronidase and
assessed immediately upon recovery for the presence of an oocyte.
Once again, no oocytes could be identified within these structures.
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There was also no evidence of a degenerate oocyte. The unusual findings with this patient are being further investigated.
This investigation on the discarded follicle-like structures found at
the time of oocyte retrieval was conducted in accordance with the
policies of the Cleveland Clinic Institutional Review Board.
Discussion
EFS remains a controversial topic, with many scientists questioning its
actual existence. The term was first used to describe the absence of
oocytes after IVF oocyte retrieval from a patient who responded
normally to follicular stimulation with rising estradiol levels and
increasing follicular diameters as measured by ultrasound (Coulam
et al., 1986). Although dysfunctional folliculogenesis has been
offered as an explanation, there is little direct evidence to explain
the etiology of this event. Other explanations offered for the
absence of oocytes have included technical difficulties during the
retrieval, premature LH surge and pharmacological problems
related to the lack of bioactive product in the hCG injection
(reviewed in Bustillo (2004)).
This case report may be the first direct evidence of the existence of
true EFS as a symptom of ovarian dysfunction. These follicle-like structures must have been prevalent in the ovarian cortex of this patient.
Upon piercing of the ovary to aspirate mature follicles, these tiny
structures were simultaneously aspirated. The presence of close to
200 pre-antral follicles in the aspirate, the presence of thin zonae
and the clear lack of oocytes are suggestive of some failure in the
oocyte maturation process. It has been previously theorized that the
absence of oocytes in EFS may be attributed to increased apoptotic
gene expression and a reduction in transcripts whose products are
responsible for healthy follicular growth. Without proper signaling, it
is postulated that the oocyte may undergo atresia (Inan et al.,
2006). This may explain our current findings.
The recurrence of these same follicle-like structures in the patient’s
second IVF attempt lends credence to the idea of genuine ovarian dysfunction rather than technical or pharmacological artifact. The in vitroplated follicles were clearly capable of growth expansion and increasing levels of estradiol secretion. Extrapolating from these data and
observations, one might hypothesize that the continued growth
within the ovary of such follicles in the absence of a viable oocyte
would ultimately result in what presents as EFS.
There have been other reports of primary and secondary follicles
being isolated from follicular aspirate, along with mature oocytes
after ovarian stimulation (Wu et al., 1998; Moskovtsev et al., 2002;
Zhang et al., 2002). However, all of these studies claim the presence
of an oocyte at the time of isolation and suggest that degeneration
occurred later, after in vitro culture for 48 h due to suboptimal
culture conditions. Our case was unique in that we found no evidence
of an oocyte even on the initial day of recovery.
The present case may be a borderline form of EFS since some
oocytes were retrieved but the number of mature ones was very
low. In EFS, the absence of oocytes may be attributed to increased
apoptosis and reduced healthy follicular growth (Inan et al., 2006).
Thus, the oocyte may have been originally present and then underwent atresia and apoptosis.
It is still a matter of debate, especially in humans, as to whether
the oocyte alone (Bousquet et al., 1981; Epifano et al., 1995) or
Desai et al.
in conjunction with GCs (Lee and Dunbar, 1993; Hinsch et al.,
1994; Sinowatz et al., 2001; Bogner et al., 2004; Gook et al.,
2004) can express and deposit the proteins needed to form the
zona. Eberspaecher et al. (2001) concluded that in mice, monkey
and humans, the zona proteins are expressed and assembled
exclusively by the oocyte and not by the GCs. They suggested
that the previous observations that showed the involvement of
GCs in zona pellucida formation may be due to improper fixation
of tissues leading to disruption of zona pellucida. If this is correct,
the presence of a thin zona in the retrieved follicle-like structures
in our present case study may be a further proof of the ovulatory
dysfunction theory, in the sense that the oocyte may have been
present and then undergone atresia after the formation of a zona
pellucida.
In summary, we report here a case of borderline EFS where ovulatory dysfunction seems to be the main cause for its pathogenesis. To
the best of our knowledge this is the first clear evidence of true empty
follicles, which we believe that, if left to grow in vivo, would have
resulted in empty Graffian follicles and genuine EFS.
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Submitted on November 25, 2008; resubmitted on December 17, 2008; accepted
on December 28, 2008