Keywords in vitro fertilization - ultrasound - ovarian function tests - ovarian reserve
Palavras-chave fertilização in vitro - ultrassonografia - testes de função ovariana - reserva ovariana
Introduction
Research on infertility has evolved with constant studies and technological advances
due to the increase of infertile couples who seek assisted reproduction (AR) services.[1 ]
[2 ] The recruitment and development of multiple ovarian follicles are key to treatment.[1 ]
[3 ]
[4 ]
The correct assessment of the ovarian reserve is a central issue in the management
of patients with infertility.[1 ]
[3 ]
[5 ] The goal is to predict the chances of response to the induction and select the “optimal”
dose for the ovarian hyperstimulation.[3 ]
[6 ]
Among the aspects of clinical history, advancing age is considered a determining factor
of fertility.[7 ]
[8 ] It causes a decrease in the ovarian reserve and an impaired oocyte quality.
The ovarian volume, thanks to its good accuracy and the cost-benefit ratio, seems
to be a useful tool in monitoring patients undergoing AR.[9 ]
Studies have demonstrated an apparent correlation between the number of antral follicles
and the functional status of the ovaries.[3 ]
[6 ]
[10 ]
[11 ]
[12 ]
[13 ] When compared, the antral follicle count (AFC) is higher than the volume of the
ovaries in the evaluation of poor response to in vitro fertilization (IVF).[7 ]
[9 ]
The basal serum follicle stimulating hormone (FSH) concentration, together with the
concentrations of estradiol,[1 ]
[14 ] represents a predictive value for ovarian response.[15 ]
[16 ] Elevated FSH levels are strongly associated with poor ovarian response, low levels
of estradiol and low rate gestation.[1 ]
[17 ] Therefore, the basal serum concentration of FSH[6 ] has been used as a marker for ovarian insufficiency.[9 ]
The dosage of anti-Müllerian hormone (AMH) has been used as an early and sensitive
marker of the ovarian reserve.[1 ]
[8 ]
[18 ] It reflects the amount of remaining primordial follicles, so this hormone is strongly
associated with the AFC.[1 ]
[8 ]
[11 ]
[19 ] The decline in AMH levels can be detected earlier than other hormonal changes and
the AFC.[1 ]
[20 ]
[21 ]
A poor ovarian response may be associated with low pregnancy rates, and cycles are
frequently cancelled without achieving the oocyte retrieval.[7 ]
[22 ]
Despite the importance of the ovarian reserve measurement, the best way to correctly
evaluate the follicular status remains controversial.[1 ] A good ovarian reserve test should be predictive of conception, and should indicate
the probable duration of ovarian activity. Furthermore, it should point out the ideal
dose of ovarian stimulation and the chance to achieve gestation successfully.[23 ]
Purpose
The main objective of this work is to identify which methods used in the assessment
of the ovarian reserve are exclusive or complementary to identify the best response
to follicle development.
Methods
After approval by the Ethics and Research Committee of the Faculdade de Medicina de
São José do Rio Preto (FAMERP) under number 18617, this study was conducted at the
Instituto de Medicina Reprodutiva e Fetal (Institute of Reproductive and Fetal Medicine
– IMR), São José do Rio Preto, São Paulo, entity convened with FAMERP. It is a retrospective
cohort study, involving patients under AR treatment for conjugal infertility.
From April 2009 to July 2014, 379 patients were submitted to ovulation induction for
high complexity AR at the IMR and selected for this work. After signing a free and
informed consent, and without prejudice to the proposed treatment, 293 patients were
included in the study.
The included patients were evaluated, regardless of their chronological age, through
biochemical tests (FSH, estradiol or AMH) and ultrasound (ovarian volume and AFC)
for analysis of the ovarian reserve.
Patients with suspicion of pregnancy; patients who would have a procedure with egg
reception; those submitted to oophorectomy and/or oophoroplasty, unilateral or bilateral;
those who underwent ultrasound in other services; the ones in which one ovary was
not visualized at baseline ultrasound; those with residual ovarian cyst (greater than
16 mm average diameter) in the moment of the first ultrasound; and the ones presenting
FSH dosages greater than 15 UI/mL were excluded.
Clinical data were collected through interviews held by the author of the work or
members of her team, including collection of medical record data.
Blood samples were drawn for FSH and estradiol dosage on the same day of the first
ultrasound, always before the beginning of the stimulus, on the third day of the menstrual
cycle at most. Anti-Müllerian hormone measurements were performed before the beginning
of the treatment, at any stage of the menstrual cycle. The ultrasound operators had
access to the laboratory tests results.
The sonographic examination was performed using the following ultrasound apparatus:
Medison, model ACCUVIX XP (Sansung, Seoul Korea); and GE, model Voluson E8 (GE Healthcare,
Austria), with an endocavitary transducer of 5–12 MHz, adequately prepared for the
implementation of endovaginal examination, performed always by the same operators
(the author of this paper and three other IMR clinical doctors), and lasting ten to
twenty minutes. The ultrasound was performed between the first and third days of the
menstrual cycle.
During the 2D ultrasound examination, the volume of the ovaries was calculated by
the measurement of their three largest diameters. The antral follicles were counted,
and they are represented at the ultrasound as small round anechoic images with diameters
between two and ten millimeters. The register was obtained by measuring the largest
diameter of each follicle. The follicle number count was performed by scanning the
ovaries transvaginally.
The new serial ultrasound was performed between six and eight days after the start
of the ovarian stimulation. From there, the ultrasound control was performed with
individual frequency for each case. Controls were performed every 2 days until reaching
follicles with a 16 mm average diameter, and daily until obtaining at least 3 follicles
of 18 mm or more for the final maturation with the human chorionic gonadotropin hormone.[10 ]
Later, the sonographic parameters were analyzed (ovarian volume, AFC and follicular
development data), as well as the age and laboratory parameters (serum levels of FSH,
estradiol and AMH), in the prediction of follicular development, and also the in the
relationships among them. Data referring to egg collection, fertilization and pregnancy
rates were not studied to avoid potential sources of bias.
The collected data were tabulated and statistically analyzed using the Statistical
Package for Social Sciences (SPPS) software for Windows (SPSS, version 20, Chicago,
US).
The association between variables was assessed using Spearman's correlation coefficient.
We used the receiver operating characteristic (ROC) curve to determine the cutoff
point, the sensitivity, the specificity and the predictive values of the tests. The
comparison between the subgroup of number of antral follicles and the ovarian volume
was analyzed by Kruskal-Wallis test. A p value of < 0.005 was considered statistically significant.
This Project did not require any financial support to be developed.
Results
Of the 293 couples included in this study, 39.6% had infertility by male factor; 50.2%
by ovarian factor; 13.7% by endometriosis; 12.3% by tubal factor; 2.7% by uterine
factor; 7.8% without factors defined and classified as infertility or sterility without
apparent cause; and 25.6% had more than 1 factor of infertility.
The average age was 34.7 years, ranging from 23 to 47 years; the FSH baseline had
a mean of 4.3 IU/mL, ranging between 0.2 and 14.6 IU/mL; the estradiol, a mean of
18.4 ng/mL; and the AMH, a mean of 1.4 ng/mL ranging between 0.3 and 3.6 ng/mL.
The ovarian volume measurement and the AFC were calculated in 293 patients, yielding
an average of 4.5 cc in volume and 7.7 in the average number of antral follicles for
the right ovary and mean of, respectively, 4.3 cc and 7.3 for the left.
Considering the age as the main variable, a significant negative correlation was observed
with the volume of both ovaries (right ovary, r = -0.21; left ovary, r = -0.22; both
p < 0.0001), the AFC (right ovary, r = -0.38; left, r = -0.47; both p < 0.0001) and the total of recruited follicles (r = -0.47 p < 0.0001).
Considering the volume of both ovaries as the main variable, a significant positive
correlation with the AFC of the respective ovary and the recruited follicles was observed,
as shown in [Table 1 ].
Table 1
Spearman Correlation between the ovarian volume and the AFC, and the number of recruited
follicles
Variable
p
AFCR
AFCL
Recruited follicles
Right ovary volume
< 0.0001
0.593
0.489
Left ovary volume
< 0.001
0.560
0.438
Abbreviations: AFC, antral follicle count; AFCL, antral follicle count in the left
ovary; AFCR, antral follicle count in the right ovary; p , percentile.
Considering the AFC as the main variable, a significant positive correlation was observed
with the total of recruited oocytes (right ovary, r = 0.73; left ovary, r = 0.72;
total antral follicles, r = 0.77; all p < 0.0001).
Considering the FSH dosage as the main variable, a significant positive correlation
with the estradiol dosage (r = 0.20; p = 0.004) was observed. This means that in patients of reproductive age, the greater
the dose of FSH, the higher the dosage of estradiol. Correlating the FSH with age
(p = 0.49; r = 0.45), a positive correlation without statistical significance was observed.
Correlating the FSH with the AFC (both ovaries, r = 0.07; p = 0.24), a positive correlation without statistical significance was obtained, probably
because patients with FSH higher than 15 UI/mL were excluded from the study. In relation
to the recruited follicles (r = -0.27; p = 0.68), a negative correlation was obtained, though not statistically significant.
The AMH versus age showed a negative correlation (r = -0.33; p = 0.21); correlating the AMH with the volume of the ovaries, positive values were
obtained (right, r = 0.49; p = 0.06 and left, r = 0.28; p = 0.30); and correlating the AMH with the AFC, positive values were obtained as well
(right ovary, r = 0.48; p = 0.06; and left ovary, r = 0.37; p = 0.17).
In making the correlation of the ovarian volume with the recruited follicles larger
than 18 mm, when using a 2.6 cm3 cutoff point in the amount of at least one ovary, we observed: a sensitivity of 81%;
a positive predictive value of 97%; and an area under the ROC curve of 0.76 ([Fig. 1 ]).
Fig. 1 Receiver operating characteristic (ROC) curve of the correlation between the ovarian
volume and the recruited follicles greater than 18 mm.
However, when we correlated the AFC with the recruited follicles larger than 18 mm,
we observed that, with a cutoff of 12 antral follicles, a positive predictive value
of 99% and an area under the ROC curve of 0.76 were obtained ([Fig. 2 ]).
Fig. 2 Receiver operating characteristic (ROC) curve of the correlation between the number
of antral follicles and the number of recruited follicles greater than 18 mm.
In making the correlation of the AFC in patients with less than three recruited follicles,
larger than 18 mm, we observed, when using a cutoff point of 11 antral follicles,
a sensitivity of 77%, a specificity of 68%, and an area under the ROC curve of 0.78.
When using a cutoff point of 5 antral follicles, a sensitivity of 26%, a specificity
of 93%, and an area under the ROC curve of 0.78. However, when we correlated the AFC
patients who had more than 15 recruited follicles larger than 18 mm, we observed,
when using a cutoff point of 15 antral follicles, a sensitivity of 97%, a specificity
of 70%, and an area under the ROC curve of 0.91.
After separating the sample into 3 groups by the number of antral follicles (< 6;
6 to 15; and > 15), we observed that the subgroup with less than 6 follicles showed
a greater age than the other groups (p < 0.0001), fewer recruited follicles than the other groups (p < 0.0001), and an FSH dosage higher than the group between 6 and 15 (p = 0.02), in which the Krustal-Vallis test for independent samples was used ([Figs. 3 ], [4 ]).
Fig. 3 Relationship between age and the number of antral follicles. Test: Kruskal-Wallis.
Fig. 4 Relationship between the number of recruited follicles and the number of antral follicles.
Test: Krustal-Wallis.
After separating the sample into 3 groups by ovarian volume (< 3 cm3 ; 3 to 10 cm3 ; and > 10 cm3 , we observed that the subgroup with volume of 3 cm3 or less presented an AFC of the respective ovary lower than the other groups (p = 0.001 for both ovaries) ([Fig. 5 ]). We could observe that even a volume lower than 3 cm3 had a total of recruited follicles (p = 0.0001 for both ovaries) lower than the other groups.
Fig. 5 A: Relationship between the number of antral follicles of the right ovary and its
respective volume. B: Relationship between the number of antral follicles of the left
ovary with its respective volume. Test: Krustal-Wallis.
Of the 293 people included in this study, 21 patients had their cycles cancelled due
to ovulation induction failure. In this group, age (p = 0.03) and FSH levels (p = 0.02) higher than in the group that completed the stimulus were observed; ovarian
volume and an AFC (p = 0.0001) lower than in the group that completed the stimulus, were also observed.
Discussion
The application of assessment tests of ovarian reserves in patients who will undergo
IVF has been widely studied, mainly due to the high cost and complexity of the treatment.
But there is still no consensus regarding the best predictor of ovarian response,
despite the fact that several studies have been published, a fact that may explain
the abundance of proposed tests.
The objective of establishing the best method or combination of these tests is to
reduce the number of tests that a particular patient should be submitted for the evaluation
of the ovarian reserve, reducing the stress and financial burden, without compromising
the necessary information to perform an IVF treatment, however.[14 ]
Considering the age as the main variable, we observed that there is an obvious and
strong correlation: the greater the age, the lower the volume of the ovaries, the
number of antral follicles, and the number of recruited follicles. The inverse association
of a woman's age with low a ovarian reserve is largely reported in other published
studies.[9 ]
[12 ]
[24 ] Choi et al[25 ] report that before the first IVF in a patient, 60% of the predicted prognostic is
for the age, and 40% for other clinical factors.
Aboulghar et al[8 ] describe that age remains as the primary determinant of success in AR cycles; however,
at any age, women with high levels of AMH have a higher success rate of oocyte recovery
after follicular puncture than those with lower levels of AMH in the same age range.
Surekha et al[26 ] confirm the data from our study when comparing the lack of a significant association
of the FSH with the ovarian reserve, unlike other specific markers, such as AMH and
AFC.
In another retrospective study, Chuang et al[27 ] concluded that the basal FSH is a good marker for the remaining follicular pool.
Magalhães et al[28 ] described that it is difficult to find associations between the FSH basal level
and the follicular count when patients with FSH levels greater than 15 UI/mL are excluded
from the study, as it was done in our work. On the other hand, Abdalla and Thum[29 ] suggest that a high rate of FSH (> 10 IU/mL) should not be a criterion for the exclusion
of patients for treatment with IVF, since the test represents a quantitative and not
qualitative aspect of the ovarian reserve, that is, despite having a low follicular
pool, one patient does not necessarily present a poor oocyte quality, especially the
ones younger than 38 years of age.[9 ]
Luna et al[17 ] describe that the cycle cancellation rates were significantly higher in patients
with elevated FSH (≥ 13.03 IU/mL) on day 3, compared with patients with normal levels
of FSH in all age groups, which was similar to the rates found in our study.
Regarding the ovarian volume, we found an association of this variable with the ovarian
response. Some studies have shown that the reduction is related to poor response to
ovarian stimulation, but the sensitivity and specificity are lower compared with the
AFC.[9 ]
[14 ]
Considering the AFC as a main variable, we found in our work that a higher AFC relates
to a greater number of recruited follicles, similar to the findings of Souza et al.[24 ] Magalhães et al[28 ] describe that the predictive power of the AFC is substantially similar to the serum
concentration of AMH, but with a higher sensitivity.[14 ] The data from our work are also similar to Barbakadze et al,[21 ] and show negative correlation between age and the AFC.
Iliodromiti et al[11 ] describe a progressive decline of AMH concentration with advancing age. Other studies
have reported a strong correlation between the number of antral follicles and the
serum basal level of AMH.[18 ] This relationship can be explained by the fact that the AMH is produced by the theca
cells of the antral follicles; therefore, the greater the number of antral follicles,
the greater the amount of hormone produced, increasing its concentration in the blood.[28 ]
Similarly to our study, Aflatoonian et al,[30 ] when correlating the AFC with the recruited follicles greater than 18 mm, observed
good sensibility and specificity (89 and 92% respectively), but used a cutoff point
of 16 antral follicles, while in our study we used 12.
Most of the bibliographic data obtained confirm that the AFC and the AMH can be used
as a screening method to detect probable poor responders, or responders with lower
reserves, and to predict the risk of ovarian hyperstimulation syndrome (OHSS) and
the cancellation of cycles, having the best predictive value of the number of oocytes
collected in IVF cycles.[1 ]
[3 ]
[7 ]
[18 ]
[19 ]
Broer et al[31 ] found a sensitivity of 82% and a specificity of 80% for a prediction of poor response.
Kwee et al[32 ] found low response with an AFC lower than 6, the same cutoff point we used, with
a sensitivity of 73% and a specificity of 95%.[1 ]
[24 ]
To predict OHSS, Aflatoonian et al[30 ] showed that the AFC and the AMH have similar accuracy, with an area under the ROC
curve of 0.961 and 0.922 respectively. Martins et al[33 ] used 20 follicles as the cutoff point, and showed that these women have an increased
risk of OHSS. In a systematic review, Nastri et al[34 ] presented that the evaluation by AFC and AMH dosage, before the ovulation induction
cycle, allows the prediction of the risk of OHSS, and that other baseline parameters,
such as age and FSH levels, are less accurate for prediction.
Although the combination of existing tests appears to be the most effective and useful
conduct for counseling infertile patients, in an attempt to provide them with some
expectations about the success of the proposed treatment,[9 ] our work shows stability in predicting ovarian reserve and subsequent oocyte recovery
after follicular puncture through a simple ultrasound routine exam used in preconception
gynecological evaluation.
Therefore, it is important to investigate the ovarian reserve through the AFC, since
it allows the optimization of treatment protocols, and a reduction of the traumatic
occurrence of cancelled cycles and of the fearful side effects of controlled stimulation
for poli-folliculogenesis, such as OHSS.[28 ]
[33 ]
Our AMH correlations were not statistically significant, probably due to the small
number of patients who collected the serum sample for this type of marker.
Cohort studies may be subject to selection bias, as in our study, in which infertility
patients submitted to AR were selected. Only women with FSH dosages lower than 15
UI/mL were included; therefore, bad potential responders were excluded and this selection
may have attenuated the overall strength of the correlations.
One aspect that still requires further research and further studies is the fact that
all existing tests, at the moment, are quantitative predictors of the follicular pool,
but are not able to evaluate the oocyte quality or the pregnancy rates.[35 ]
Conclusions
We concluded that age and the AFC are effective predictors of ovarian response in
AR cycles. The ovarian volume and the AMH dosage also appear to be suitable markers
of ovarian reserve.
Future Perspectives
The AFC, when performed by the three-dimensional method, appears to offer advantages
over the two-dimensional method, among which we mention: the ability to produce images
in three different planes; the possibility of virtual coloration of the follicles,
eliminating the need to repeat the process or the failure to identify them, thus eliminating
the technical issue that the AFC is operator-dependent[19 ]
[26 ]; and greater speed of the test execution and greater comfort for the patients, since
the obtained images can be analyzed later.[28 ]
The 3D ultrasound with sonography-based automated volume count (SonoAVC, GE Healthcare,
Austria) is used to obtain more precision and better reproduction;[14 ] however, early studies show no statistical difference between the two and three-dimensional
evaluations.
In our service, this technique is already being used, and, in the future, we can compare
our data (two-dimensional evaluation) to study the correlation of the three-dimensional
evaluation with the serum levels of AMH, the randomization of ovarian hyperstimulation
protocols, with consequent follicular response, and the dopplerfluxometric analysis
of oocyte quality, results that could help to achieve better pregnancy outcomes.
