Objective: The study aims to assess the effect of laparoscopic ovarian drilling (LOD) in clomiphene citrate
resistant patients with polycystic ovary syndrome (PCO) on the changes occurred in ovarian reserve.
Study design: A prospective cohort study.
Setting: Sohag University Hospital, Sohag, Egypt.
Materials and methods: Thirty-seven patients with PCO were enrolled in the study. We evaluated the
effect of LOD on the ovarian reserve using hormonal assay (measuring FSH, LH, AMH, Testosterone,
SHBG, E2 and FAI) and sonographic markers (including ovarian volume, antral follicle count (AFC) and
ovarian stromal blood flow).
Results: Amongst the 37 patients underwent LOD, regulation of the cycle occurred in 20 patients after
6 months (54.06%) (p = 0.001). Occurrence of pregnancy occurred in 4 patients after three months and
in 18 patients after 6 months. As regard the hormonal profile, LOD has reducing effect on the serum levels of FSH, LH, AMH, SHBG, Testosterone and free androgen index. This reduction is of statistically significance in case of LH, AMH, testosterone and FAI especially after 6 months (p = 0.01, 0.001, 0.05, 0.05 respectively). The deleterious effect of LOD was statistically significant in the AFC which was reduced
from 18.1 ± 2.7 to 11.5 ± 1.8 after 6 months (p = 0.05). Also, the ovarian blood flow indices were reduced with p = 0.01 for all indices.
Conclusions: Using the AMH and AFC as reliable markers of the ovarian reserve and measuring them for
women with anovulatory PCO undergoing LOD may provide a useful tool in evaluating the outcome of
LOD as the gold standard treatment for CC resistant PCO women.
- Introduction
Polycystic ovary (PCO) is considered as one of the most
common endocrine disorder that occurred in 5–10% of women in
reproductive age group. It is the major cause of ovulation – related
infertility, accounting for at least 75% of cases with anovulatory
infertility [1].
This major disorder is characterized by a marked increase in
preantral follicular number arranged peripherally around a dense
core of stroma or scattered throughout an increased amount of
stroma [2]. The first line of treatment of such disorder was
induction of ovulation using clomiphene citrate (CC) and other
members of selective estrogen receptor modulators (SERM) [3].
However; 15–40% of patients with CC – resistant PCO patients
[4]. Such patients can be managed either by using gonadotropins
[5] or by minimal surgical procedure known as laparoscopic ovarian
drilling [6].
The mechanism of action of LOD is largely unexplained. In particular,
it is not known whether LOD exerts its action through a
direct effect on the ovary or through a systemic endocrine mechanism
[7]. One of the theories that explain the effect of LOD is the
higher ovarian reserve presents in patients with PCO. This proposed
mechanism of exaggerated ovarian activity (at supraphysiological
state) can be reduced to the physiological level by
destruction or removal of some part of the ovary retaining the
ovarian activity to physiological state [8]. - Although the available data in the literature is limited, there is
no concrete evidence of a diminished ovarian reserve or premature
ovarian failure due to LOD in patients with PCOS. Most of the
changes in the ovarian reserve markers observed after LOD could
be interpreted as normalization of ovarian function rather than
reduction in ovarian reserve [8,9].
Anti-mullerian hormone (AMH) or mullerian inhibiting substance
(MIS) is a member of transforming growth factor b (TGF –
b). It is a dimeric glycoprotein which is produced by the granulosa
cells of primary, pre-antral and small antral follicles suggesting an
important role of AMH in human folliculogenesis [10]. AMH has
been shown to lower the sensitivity of follicles to circulating FSH
[11]. Its principle function is the inhibition of primordial follicle
growth organization that is important in dominant follicle selection
[12]. It has been shown that the serum level of AMH is likely
to be 2–3 folds increased in serum from women with PCO than in
women with healthy ovaries [13].
The aim of our study is to assess the short term effect of LOD as
regard its effect on ovarian reserve (evaluated by assessing the
sonographic parameters including AFC, ovarian volume and ovarian
stromal blood flow and hormonal parameters including AMH,
baseline FSH, LH, E2 and FSH/LH ratio and finally ovulation
monitoring.
2. Materials and methods
This is a prospective cohort study included 37 infertile anovulatory
women with CC-PCO who underwent LOD. The study was
conducted in Sohag University Hospital, OB/GYN department, on
attendants of outpatient clinic complaining from infertility during
the period from September 2015 till October 2016.
The patients were selected as infertile patients with PCO who
were resistant to CC induction of ovulation. The patient ages range
between 19 and 30 years. The age and the body mass index were
recorded for all members of study group. CC resistance was defined
as failure of ovulation after six successive cycles of ovulation
induction using a maximum dose of clomiphene citrate
(200 mg/d) for 5 days starting from day 2 to day 6 of the cycle.
PCO women were diagnosed according to the 2003 ESHRE/
ASRM (Rotterdam) criteria and their partners had normal semen
analysis (according to WHO criteria). The Rotterdam criteria for
diagnosis of PCO include presence of at least 2 out of 3 of the
following criteria: oligomenorrhea or amenorrhea, clinical and biochemical
signs of hyperandrogenism and finally ultrasonographic
picture of PCO (12 subcortical follicles (2–9 mm in diameter)
with dense stroma and/or increased ovarian volume (more than
10 cm3).
We excluded infertile patients due to causes other than PCO
(tubal block, abnormal semenogram, etc.), patients with any
organic pelvic disease diagnosed during laparoscopy or diseases
potentially affecting the normal ovarian functions as endometriosis
and fibroid, hose with previous pelvic surgery (appendectomy,
ectopic pregnancy, ovarian cystectomy, myomectomy, those with
medical diseases that may affect fertility as diabetes mellitus, liver
diseases. Additionally women with hyperprolactinemia or other
endocrine disorders that may affect fertility were excluded from
the study. Women suffering from other causes of hyperandrogenism
as congenital adrenal hyperplasia, androgen secreting
tumors and Cushing’s syndrome were also excluded.
All participants were subjected to full detailed history including
menstrual history, medical history, surgical history, past history,
obstetric history or sexual history and any previous investigations
done or treatment given. Careful general, abdominal and pelvic
examination was done and completing the investigations was
carried out. All women gave their written informed consent and
agreed to undergo the laparoscopic procedure.
Ultrasonography was performed using transvaginal sonography
probe 7.5 mHz (Sonoscape S11, Sonoscape Co Ltd. Beijing, China) to
confirm the diagnosis of PCO, to assess the ovarian volume, to
exclude ovarian or adnexal pathology and to assess the mean
AFC in both ovaries (measuring 2–9 mm). Power Doppler measurements
were performed on the early follicular phase before LOD and
repeated 3 and 6 months after LOD (also in early follicular phase).
The ovarian volume was measured using the following formula
(length width thickness 0.523). The procedure was done on
the two ovaries and the summation of volumes of both ovaries
was calculated giving the total ovarian volume (TOV).
Antral follicles were defined as every hypo echoic rounded
structures measuring 2–10 mm seen subcapsular or within the
ovarian stroma. AFC defined as the count of all antral follicles measuring
2–10 mm in both ovaries at the baseline examination session
(early follicular phase). Serial scans were obtained by slow
sweeping of the transvaginal probe from medial to lateral border
of the ovary in two perpendicular planes. The procedure was performed
on the other ovary to obtain the total antral follicle count.
The ovarian stromal blood flow measurement was achieved
using 3D power Doppler ultrasound (Sonoscape S40, Sonoscape
Co Ltd. Beijing, China). Flow velocimetry waveforms were obtained
from stromal blood vessels away from the ovarian capsule. The
gate of Doppler apparatus was positioned when the vessels with
good color signals was identified on the screen.
The built in VOCAL (Virtual Organ Computer – aided Analysis)
imaging program of the 3 power Doppler histogram analysis was
used to determine the ovarian volume as well as the vascularization
and blood flow indices. During the analysis and calculation,
the manual mode of VOCAL contour editor was used to cover the
whole 3D volume and the ovary with a 15 degree rotation step.
Hence, 12 contour planes were analyzed for each ovary to cover
180 degree. Vascularization index (VI), Flow index (FI) and Vascularization
flow index (VFI) were measured.
Blood samples were collected before LOD and after 3 and
6 months after LOD to measure plasma concentrations of E2, FSH,
LH, AMH, SHBG and testosterone. Before surgery, about 5 cc blood
sample was taken from each patient and maintained in tubes containing
cloth activator material (serum separation, Stago, France).
The samples were centrifuged with 3000 rpm and the serum was
collected at 2 ml micro tubes and stored at –20 C freezer until
subsequent analysis. Congenital adrenal hyperplasia was excluded
with a single measurement of serum 17-hydroxyprogesterone (17-
OHP) levels (normal value < 1.98 ug/l). Hyperprolactinemia was
excluded with a single assay of serum prolactin (PRL) levels (normal
values < 25 ng/ml).
Plasma samples were assayed for the hormones in duplicate
using a commercial enzyme-linked immunosorbant assay kit (Pritest
ECO, ELISA, ROBOnik (India) Pvt, Ltd) according to the manufacturer’s
protocol. The sensitivity of the assay was 0.24 ng/ml. The
intra- and inter-assay variability were <5% and 8%, respectively.
In each woman, the free androgen index (FAI) was calculated using
the following formula: FAI = 100 Total Testosterone/SHBG.
Using the standard precautions used to be followed in laparoscopy,
with the patient put in lithotomy position ovarian drilling
was done under general anesthesia. After Co2 insufflation of the
abdomen using Verres needle the patient was put in moderate
Trendelenburg position (30 degrees) then a 10-mm trocar inserted
intraperitoneally. The trocar sleeve was left in situ and a 10-mm 0
degree telescope (KARL STORZ co Ltd, Arizona, United States) was
inserted and connected to a camera with a video monitor system.
A panoramic view was performed before making incisions for
instrumentation. The abdominal cavity especially the pelvic area
was carefully inspected. Two further 5-mm trocars were inserted - through small incisions in each of the iliac fossae, one for a grasping
forceps and the other for a bipolar diathermy electrode. The
ovary was stabilized by grasping the ovarian ligament with a
grasping forceps, the ovary was left away from the bowel to avoid
pelvic organ injuries. The ovarian cortex was pierced to a depth of
6 mm using a bipolar electrode set to a power of 30–50Wand cauterized
for 2–4 s. The number of punctures made depended on the
size of the ovary and number of subcapsular cysts visible at
laparoscopy.
All punctures were done on the antimesenteric border to protect
against damage to the ovarian blood supply. Adequate care
was taken to avoid overly aggressive drilling, which may lead to
excessive tissue destruction, adhesion formation, and even ovarian
failure. At the completion of the procedure, 200 ml of normal saline
was then introduced into the pouch of Douglas in order to enhance
ovarian cooling after diathermy.
All the patients were followed up for 6 months. The main outcome
measures included: Clinical outcomes as menstrual pattern,
ovulation rate and occurrence of pregnancy 3 and 6 months after
LOD. Ovarian reserve assessment: either through hormonal profile
or through sonographic evaluation.
2.1. Statistical analysis
Statistical analysis was done using SPSS software version 10.
Data were presented as mean ± SD for parametric variables and
as number and percent for non – parametric variables. For comparing
of parametric data, unpaired t-test was used to compare
between 2 independent parametric groups. Mann-Whitney test
was used to compare between 2 independent non – parametric
groups. Comparison of categorical data was carried out using X2
(Chi – square test). P-value < 0.05 was considered statistically
significant.
3. Results
The study included 37 patients who were fulfilled the inclusion
criteria. The baseline characteristics and basal hormonal profile
levels were summarized in Table 1. Additionally, Table 2 shows
the baseline ovarian volume, AFC, and 3D power Doppler indices
of the ovarian stromal blood flow at inclusion.
Table 3 demonstrates the clinical outcomes of the study
participants 3 and 6 months after LOD. Amongst the 37 patients
underwent LOD, regulation of the cycle occurred in 6 patients after - 3 months (16.22%) and in 20 patients after 6 months (54.06%)
(p = 0.05 and 0.001 respectively). Occurrence of pregnancy
occurred in 4 patients after three months [1 patient (2.71%) spontaneously
and 3 patients (8.11%) after induction] and in 18 patients
after 6 months [7 patients (18.91%) spontaneously and 11 patients
(29.7%) after induction].
As regard the hormonal profile, LOD has reducing effect on the
serum levels of FSH, LH, AMH, SHBG, Testosterone and free androgen
index. This reduction is of statistically significance in case of
LH, AMH, testosterone and FAI especially after 6 months
(p = 0.01, 0.001, 0.05, 0.05 respectively) (Table 4).
The deleterious effect of LOD was statistically significant in the
AFC which was reduced from 18.1 ± 2.7 to 11.5 ± 1.8 after
6 months (p = 0.05). Also, the ovarian blood flow indices were
reduced with p = 0.01 for VI, FI and VFI (Table 5).
4. Discussion
Risk and benefit of any modalities of treatment of infertility
should be categorized accordingly. In patients with PCO who are
diagnosed as CC resistant, the treatment of infertility is achieved
either medically (by using gonadotropin) or surgically (through
LOD). Despite being minimally invasive, LOD is considered as one
of the cornerstone in treatment of PCO-related infertility. However,
the procedure is not free from certain effects on ovarian reserve
and occurrence of pelvic adhesions.
Amongst the 37 patients with CC resistant PCO, regulation of
the cycle was occurred in 6/37 (16.22%) of patients 3 months after
LOD and this ratio increased to 20/37 (54.06%) of patients after
6 months. On the other hand, occurrence of pregnancy occurred
in 4/37 (10.81%) and 18/37 (48.65%) of patients 3 months and
6 months after LOD respectively. These results are in agreement
of previous study done by Poujade et al. who did their research
on 74 patients with CC resistant PCO and found occurrence of pregnancy
47/74 (63%) of patients but within 11 month after LOD [14].
In our study, at the end of 6 month observation after LOD, 19/37
(51.35%) of patients remained infertile and could not conceive even
after regulation of the cycle either spontaneously or after using
ovulation induction. This is can be explained by either presence
of other subtle causes as hyperprolactinemia, subtle anatomical
cause as subseptate uterus, development of male factor e.g. development
of varicocele or even due to development of coital problem.
Another cause may be attributed to insufficient drilling
enough to induce favorable changes on reproductive parameters.
As regard the hormonal profile changes, LOD had variable effects
on different hormones. E2 showed slight increase in 3 and 6 months
after LOD, but with no statistical significance. Also the present
study showed minimal reducing changes in the serum level of
FSH. These changes are statistically insignificant. These findings
are matched with that of Kucuk and Kilic-Okman [15] who did his
experiment on 22 nulliparous oligomenorrhic women and found
no difference in E2 or FSH level in the early follicular phase in the
early postoperative period (with p = 0.255, p = 0.14 respectively).
In contrast to FSH, there were obvious changes of statistical
significance in the serum level of LH. Our study showed marked - reduction in the serum level of LH which was reflected on
FSH / LH ratio in 3 months and 6 months after LOD. These results
were agreed with those of Al-Ojaimi, 2004 who did his research on
181 patients in Bahrain. He found a significant decline in LH, LHFSH
ratio and testosterone with a significant increase in FSH [16].
The serum level of testosterone showed statistically significant
reduction when compared from pre LOD and during the three and
six months follow–up period after LOD (from 2.61 ± 1.15 pre-LOD
to 1.61 ± 0.6 and 1.43 ± 0.51 three and six months post -LOD
respectively). This significant reduction also occurred in free
androgen index (from 5.75 ± 0.9 pre-LOD to 3.13 ± 0.8 and
2.9 ± 0.71 three and six months post-LOD). This finding is coincide
with those obtained by Sunj et al., 2014 whom results obtained
from 96 CC resistant PCO patients revealed marked reduction of
AMH level in the serum in good responders to LOD [17].
AMH is considered as a newest and the most predictive marker
of ovarian reserve [18]. AMH may constitute a marker of ovarian
aging as it correlates with the number of early antral follicles
which might – in turn – represent the size of resting pool of follicles
[19]. Therefore, serum AMH levels are affected by reduced
number of antral follicles or ovarian tissue injuries [20]. Studies
showed that estimation of serum AMH revealed significant higher
levels of such hormone in the plasma of women with anovulatory
PCO that could be explained by excessive production of AMH by
the increased number of early antral follicles [21].
Our study showed a marked change in the serum level of AMH
with statistically significant reduction of the serum level of such
hormone from 6.3 ± 2.1 ng/ml pre-LOD to 4.1 ± 1.8 ng/ml
(3 months after LOD) and 3.9 ± 1.51 ng/ml (6 months after LOD).
These results are consistent with those of Weerakiet et al. who
estimated the serum level in 21 PCO patients before and after
LOD and found significant reduction comparable to our results
[22]. However, our results are not matched with those of Farzadi
et al. [23] who applied his research on 30 patients with PCO and found that LOD didn’t change anti-mullerian hormone serum
(8.4 ± 4.7 before laparoscopic surgery and 7 ± 4.5, and
7.7 ± 4.4 ng/ml 3 and 6 months after surgery, respectively and
hence has no adverse effects on the ovarian reserve.
Considering the AFC, it is universally accepted that AFC is one of
the most discriminative test for evaluation of ovarian reserve, and it
has different advantages being easy to be done, non invasive test
and is considered cheap in comparison to other ovarian reserve
tests. However, one of the most common draw backs of measuring
AFC is being subjected to assay variations due to intra and inter
observer differences and require additional time and manpower
to be performed.
Our study showed significant reduction in the AFC. Preoperative,
the mean AFC in our study was 18.5 ± 2.6; this number is
markedly reduced to be 12.1 ± 1.9 three months after LOD and persisted
low to be 11.5 ± 1.8 after 6 months of LOD. These noticeable
changes could be explained by the direct thermal effect of LOD on
the ovarian stromal tissue with subsequent decrease in AFC. Our
results are in agreement with those of Ashraf, 2011 [24] study
which showed marked reduction in AFC after LOD from
29.0 ± 2.4 pre-operative to 15.0 ± 2.2 post LOD. Again, the significant
reduction in the serum level of AMH and AFC could be
explained by the possible ovarian parynchematous and ovarian
vasculature damage after use of diathermy.
Studying the ovarian stromal blood flow in PCO patients in our
study showed statistically significant reduction in VI, FI and VFI
after LOD. These results are in agreement with those of Ashraf,
2011 [24] whose results showed significant reduction in ovarian
stromal blood flow, where the VI decreased from 4.8 ± 1.3 pre-
LOD to be 2.4 ± 0.75 after LOD. The same could be applied on FI
which decreased from 52.4 ± 4.3 pre-LOD to 44.3 ± 2.5 after LOD
and VFI which also decreased from 2.9 ± 0.43 to 1.2 ± 0.59 pre
and after LOD respectively. It is notably mention that the high
ovarian stromal blood flow in patients with PCO could be explained
by the high vascular endothelial growth factor secondary to high
LH level in PCO patients. Thus thermal reduction of LH – producing
ovarian parynchematous tissue which in turn reduces the serum
level of VEGF and IGF-1 can be the causes that reduce the ovarian
stromal blood flow after LOD.
In conclusion, the present study demonstrates that significant
high levels of plasma AMH in women with anovulatory PCO compared
with healthy controls. 3D power Doppler ovarian stromal VI,
FI, and VFI were significantly higher in women with PCOS than in
healthy fertile women. Measuring AMH and ovarian stromal 3D
power Doppler blood flow for women with anovulatory PCO
undergoing LOD may provide a useful tool in evaluating the outcome
of LOD. Further clinical studies are needed to clarify the
underlying mechanisms controlling ovarian stromal blood flow
and AMH. LOD had appeared after the study not to be associated
with an increased risk of diminished ovarian reserve. Most of the
changes in the ovarian reserve markers raised with the current
work after LOD could be interpreted with the normalization of
ovarian function in the enrolled PCO women rather than the reduction
of the ovarian reserve.
It can be recommended that using the AMH and AFC as reliable
markers of the ovarian reserve and measuring them for women
with anovulatory PCO undergoing LOD may provide a useful tool
in evaluating the outcome of LOD as the gold standard treatment
for CC resistant PCO women.