This article is protected by copyright. All rights reserved

The assessment of ovarian reserve by antral follicle count in ovaries with

endometrioma

Maria Lúcia Lima1; Wellington P Martins1; Marcela A. Coelho Neto1; Carolina O Nastri1,2; Rui A

Ferriani1; Paula A Navarro1

1. Department of Obstetrics and Gynecology, Medical School of Ribeirao Preto, University of Sao

Paulo, Ribeirao Preto, Brazil; 2. School of Health Technology – Ultrasonography School of Ribeirao

Preto, Ribeirao Preto, Brazil.

Contact authors: Wellington P. Martins. Av. Bandeirantes, 3900 – 8 andar - HCRP - Campus

Universitario, Ribeirao Preto, Sao Paulo, Brazil, 14048-900. Phone: +55(16)3602-2583; Fax:

+55(16)3633-0946; E-mail: wpmartins@gmail.com.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/uog.14763

This article is protected by copyright. All rights reserved

Abstract

Objectives: To evaluate whether the antral follicle count (AFC) is underestimated in the presence

of an endometrioma.

Methods: Retrospective cohort study assessing all women undergoing IVF/ICSI between January

2011 and December 2012 who had both ovaries and unilateral endometrioma. The primary endpoint

was the difference between AFC and the number of oocytes retrieved per ovary.

Results Within the study period, 787 women underwent IVF/ICSI in our clinic. Sixty of these

women had at least one endometrioma; but 23 women were not included in the analysis: six had

only one ovary and 17 had bilateral endometriomas. Therefore 37 women were included and

analysed in this study and their main characteristics were: age = 34.8 ± 5.6 years (mean ± SD), BMI =

23.2 ± 4.5 kg/m², mean diameter of the endometriomas = 19.9 ± 10.7 mm. Compared with the

contralateral ovaries, the ovaries with endometrioma were larger (10.3cm³ [4.7; 18.9] vs. 3.6cm³

[2.7; 7.5], P<0.001; median [interquartile range]), and presented a lower AFC (3.0 [1.0; 6.0] vs. 5.0

[2.0; 6.5], P=0.001). However, the number of oocytes retrieved was similar between ovaries with

endometrioma (2.0 oocytes [0.5; 5.0]) and the contralateral ovaries (2.0 oocytes [0.0; 4.0], P=0.60).

Accordingly, the difference between AFC and the number of oocytes retrieved was smaller in the

ovaries with endometrioma (0.0 oocytes (-1.0; 1.5) vs. 2.0 oocytes (0.0; 4.0), P=0.005).

Conclusions: Although the AFC is reduced in ovaries with endometrioma, the number of oocytes

retrieved is similar; suggesting that the AFC is underestimated in such ovaries. We believe that this is

consequence of an impaired ability to detect small follicles in the presence of an endometrioma.

This article is protected by copyright. All rights reserved

Introduction

Laparoscopic removal of endometriomas has been associated with a negative impact on

reproductive life highlighted by the occurrence of menopause at a younger age 1 and higher rate of

premature ovarian failure 1, 2. However, the effect of surgery for endometriomas on the ovarian

reserve is controversial. In one hand, anti-Müllerian hormone (AMH) decreases after surgery 3, 4 and

the magnitude of the decline is greater for those women with bilateral ovarian surgery 4. On the

other hand, antral follicle count (AFC) of the ovary with endometrioma does not seem to change

before and after the surgery 5.

AMH and ACF are currently the two most commonly used methods to access ovarian functional

reserve 6, 7. AMH is a glycoprotein produced by granulosa cells from pre-antral and antral follicles

that is secreted into circulation; in this way, serum AMH reflects the combined follicular pool from

both ovaries. Serum AMH measurements are made through three commercially available kits and,

although they show a good correlation, the absolute values may vary by approximately 30% 8. For

AFC estimation, each ovary is directly identified by two or three-dimensional ultrasonography and

follicles between 2 and 10 mm are counted. Although this technique is dependent on image quality

and observer expertise, AFC measurement shows a sufficient inter-observer reproducibility 9, and it

allows assessing each ovary separately.

One must be aware of other limitations of the ultrasonography. The presence of the

endometrioma and its local inflammation might reduce follicle count 10, but also distort the adjacent

anatomy and to greater distance between the normal ovarian tissue and the probe might increase

the attenuation and impair the identification of antral follicles 11-13. Hence, ovarian reserve could be

underestimated in the presence of endometrioma, which is less likely after the surgery, when the

pelvic anatomy is closer to the physiologic. These could explain the discrepancy observed between

AMH and AFC evaluated before and after laparoscopy for endometriomas: the reduction in ovarian

reserve caused by surgery might be compensated by an improved ability to identify small follicles

This article is protected by copyright. All rights reserved

when assessing ovarian reserve by AFC 14.

The aim of this study was to answer the question: is the assessment of ovarian reserve by AFC in

ovaries with endometrioma underestimated? We thereby evaluated the difference between AFC

and number of oocytes retrieved comparing the ovaries with and without endometrioma from

women with unilateral endometrioma undergoing controlled ovarian stimulation (COS) for IVF/ICSI.

Methods

Study design

We performed a retrospective cohort study assessing all women undergoing IVF/ICSI at the

fertility clinic of the university hospital of the Medical School of Ribeirao Preto, University of Sao

Paulo, Brazil, between January 2011 and December 2012. Data was collected from medical records

by one author (MACN) within June 2013 and February 2014. The study was approved by the

Institutional Review Board; and no additional consent was requested from the participants.

Participants

All women who underwent COS for IVF/ICSI in the study period and had both ovaries and

unilateral endometrioma were considered eligible; only data from the first cycle of COS of each

woman during the study period was included in the analysis. Women were followed up until follicle

aspiration for oocyte retrieval. Data from all included women were analysed.

Variables and data sources

All data were obtained from medical records. For all the assessed parameters, the unit of analysis

was the individual ovary – each woman contributed with two ovaries, one with and one without

endometrioma. The primary endpoint was the difference between AFC and the number of oocytes

retrieved per ovary. The number of oocytes retrieved per ovary is the most robust measurement of

ovarian reserve and AFC should be a good predictor of this number; therefore, if the difference

This article is protected by copyright. All rights reserved

between AFC and the number of oocytes retrieved (AFC - oocytes retrieved) were larger in the

ovaries without endometrioma, it suggests that AFC is possibly underestimated in ovaries with

endometrioma. The following parameters were assessed: age (years), weight (kg), height (m), body

mass index (BMI, kg/m²), the maximum diameter of the endometrioma, and ovarian volume.

Bias

To avoid selection bias we considered eligible all women starting an IVF/ICSI cycle with both

ovaries and unilateral endometrioma during the period of enrolment. When a woman underwent

more than one cycle during the study period we only included data from the first cycle. All included

women received hCG and underwent oocyte retrieval; there were no missing data.

Analysis and Statistics

The number of woman undergoing IVF/ICSI with unilateral endometrioma during the study

period determined the sample size. Normal distribution of the evaluated parameters was examined

by D’Agostino-Pearson omnibus normality test. All statistical analyses were performed using Prism

(Version 6.0 for Windows, GraphPad Software Inc., La Jolla, CA, USA) by one of the authors (WPM).

The evaluated parameters were described as either mean and standard deviation (SD) or median

and interquartile range, depending on the normality distribution. The following parameters were

compared between ovaries with and without endometrioma: ovarian volume; AFC; number of

oocytes retrieved; and the difference between AFC and the number of oocytes retrieved. These

outcomes were compared by Wilcoxon matched-pairs signed rank test.

Results

Participants

Within the study period, 787 women underwent IVF/ICSI in our clinic. Sixty of these women had

at least one endometrioma; but 23 women were not included in the analysis: six had only one ovary

and 17 had bilateral endometriomas. Therefore 37 women were included and analysed in this study.

This article is protected by copyright. All rights reserved

None of them had been submitted to previous surgery for endometrioma. All these 37 women

received hCG for triggering final follicular maturation and underwent oocyte retrieval.

Descriptive Data

The main characteristics of the included women were: age = 34.8 ± 5.6 years (mean ± SD), range =

22 to 48 years; BMI = 23.2 ± 4.5 kg/m², range = 17.8 to 36.9 kg/m². Down regulation was performed

with a standard long protocol starting GnRH agonist in the mid luteal phase of the previous cycle in

27 women; in the other 10 women, down regulation was performed in a flexible GnRH antagonist

protocol, starting the antagonist on the day the diameter of the largest follicle was ≥ 14 mm. The

average total dose of FSH was 1,977 ± 703 IU, range = 1,050 to 3,600. Roughly 45% of the included

women (17/37) had endometrioma in the right ovary and 54% (20/37) in the left ovary. The mean

diameter of the endometriomas was 19.9 ± 10.7 mm, range from 7 to 46 mm.

Main results

All comparisons between ovaries with and without endometriomas are presented in Table 1.

Ovaries with endometrioma were larger, presented lower AFC and similar number of oocytes

retrieved when compared with ovaries without endometrioma, in both parametric and con-

parametric comparison. Accordingly, the difference between AFC and the number of oocytes

retrieved was smaller in the ovaries with endometrioma (Table 1, Figure 1).

Discussion

Key Results

We found that the assessment of ovarian reserve by AFC in ovaries with endometrioma is

probably underestimated, because we observed a higher number of oocytes retrieved than expected

by assessing AFC in the ovaries with endometrioma, considering both ovaries.

This article is protected by copyright. All rights reserved

Limitations

As this is a retrospective cohort, we had to rely on information collected and registered in the

medical records. Another issue was the relatively small sample size due to very restrictive eligibility

criteria: we only included women with ovaries, unilateral endometrioma, and only the data from

their first cycle - to avoid including the same woman more than once. Other limitation of this study

was not to assess whether the removal of the endometrioma is able to restore the ultrasound ability

to detect small follicles.

Interpretation

The endometrioma is filled with endometrial tissue in which internal fluid is generated from the

accumulation of menstrual debris from the shedding of the active implants inside the cyst. The

inflammation caused by recurrent haemorrhage induces cell proliferation through a cytokine

cascade and production of reactive oxygen species 15. Although available evidence don’t suggest that

pregnancy and delivery rates are reduced in women with endometriosis 20, the establishment of this

local proinflammatory microenvironment damages the surrounding tissue with an establishment of

vasculature through angiogenesis, vasodilation and congestion derived from inflammatory liquid,

and substitution of normal ovarian cortical tissue for fibrous tissue 15.

This agglomeration of liquid and inflammatory cells generates a mechanical stretch of the ovary,

distorting the pelvic anatomy 16 a and increasing the distance from the probe to the ovary. The larger

propagation path of the acoustic wave reduces its intensity 11, 12. The resulting attenuation is the

consequence of reflection and scattering and also of friction-like losses induced by oscillatory tissue

motion produced, which converts the original mechanical energy into heat. This energy loss to

localized heating is referred to as absorption and is the most important contributor to ultrasound

attenuation 17. Longer path length and higher frequency waves result in greater attenuation which

impairs the resolution of the transvaginal ultrasound scan reducing the ability to detect small

follicles. After the removal of the endometrioma, it is possible that the ultrasound probe would be

This article is protected by copyright. All rights reserved

able to get closer to the ovaries, leading to a more accurate AFC.

In a recent meta-analysis Muzii et al 5 concluded that the surgical treatment for the

ovarian endometrioma does not affect the ovarian reserve because the AFC is not reduced after

surgery when compared to pre-surgery values. Their conclusion does not explain the conflicting

outcomes found for AMH and AFC: laparoscopic striping of endometrioma does not seem to

reduce ovarian reserve examined by AFC 5 whilst there is evidence that the same intervention

impairs ovarian reserve assessed by AMH 3, 4, 18, 19. The authors addressed this question,

hypothesizing that AFC would be better than AMH to evaluate ovarian reserve when considering

surgery for ovarian endometrioma, because it takes into account the laterality of the ovary,

being able to examine the impact of the surgery only in the ovaries submitted to the

intervention. However, considering the present study, we believe that the presence of ovarian

endometrioma would lead to a reduced ability to identify small follicles by transvaginal

ultrasound, and such problem would be at least partially solved by the surgical excision of the

endometrioma. Considering all these, the surgery for endometrioma might reduce ovarian

reserve even if no difference is observed in AFC, because it also improves the ability of the

transvaginal ultrasound scan to detect smaller follicles.

Implications to clinical practice and future research

Although the AFC is reduced in ovaries with endometrioma, the number of oocytes retrieved is

similar; suggesting that the AFC is underestimated in such ovaries. We believe that this is secondary

to an impaired ability to detect small follicles in the presence of an endometrioma, but further

studies with larger samples are needed to confirm our findings. Additionally, we believe to be

interesting to examine the difference between the AFC and the number of retrieved oocytes in

ovaries submitted to surgical procedures to evaluate whether the surgical stripping of

endometrioma restores the ability to detect small follicles.

This article is protected by copyright. All rights reserved

Acknowledgments

The authors would like to thank the staff of the Laboratory of Assisted Reproduction, particularly

Sandra Aparecida Cavichiollo, Marisa Blanco, Maria Cristina Picinato, and Ricardo Perussi e Silva for

technical support. Authors received salary from their institutions and were funded by two Brazilian

official agencies: CNPq and FAEPA. The funders had no role in study design, data collection and

analysis, decision to publish, or preparation of the manuscript.

References

1. Coccia ME, Rizzello F, Mariani G, Bulletti C, Palagiano A, Scarselli G. Ovarian surgery for

bilateral endometriomas influences age at menopause. Hum Reprod 2011; 26: 3000-3007.

2. Busacca M, Riparini J, Somigliana E, Oggioni G, Izzo S, Vignali M, Candiani M. Postsurgical

ovarian failure after laparoscopic excision of bilateral endometriomas. Am J Obstet Gynecol 2006;

195: 421-425.

3. Raffi F, Metwally M, Amer S. The impact of excision of ovarian endometrioma on ovarian

reserve: a systematic review and meta-analysis. J Clin Endocrinol Metab 2012; 97: 3146-3154.

4. Somigliana E, Berlanda N, Benaglia L, Vigano P, Vercellini P, Fedele L. Surgical excision of

endometriomas and ovarian reserve: a systematic review on serum antimullerian hormone level

modifications. Fertil Steril 2012; 98: 1531-1538.

5. Muzii L, Di Tucci C, Di Feliciantonio M, Marchetti C, Perniola G, Panici PB. The effect of

surgery for endometrioma on ovarian reserve evaluated by antral follicle count: a systematic review

and meta-analysis. Hum Reprod 2014; 29: 2190-2198.

6. Hendriks DJ, Mol BW, Bancsi LF, Te Velde ER, Broekmans FJ. Antral follicle count in the

prediction of poor ovarian response and pregnancy after in vitro fertilization: a meta-analysis and

comparison with basal follicle-stimulating hormone level. Fertil Steril 2005; 83: 291-301.

This article is protected by copyright. All rights reserved

7. La Marca A, Sighinolfi G, Radi D, Argento C, Baraldi E, Artenisio AC, Stabile G, Volpe A. Anti-

Mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum

Reprod Update 2010; 16: 113-130.

8. Nastri CO, Teixeira DM, Moroni RM, Leitao VM, Martins WP. Ovarian hyperstimulation

syndrome: physiopathology, staging, prediction and prevention. Ultrasound Obstet Gynecol 2014; In

press.

9. Jayaprakasan K, Walker KF, Clewes JS, Johnson IR, Raine-Fenning NJ. The interobserver

reliability of off-line antral follicle counts made from stored three-dimensional ultrasound data: a

comparative study of different measurement techniques. Ultrasound Obstet Gynecol 2007; 29: 335-

341.

10. Kitajima M, Dolmans MM, Donnez O, Masuzaki H, Soares M, Donnez J. Enhanced follicular

recruitment and atresia in cortex derived from ovaries with endometriomas. Fertil Steril 2014; 101:

1031-1037.

11. Riccabona M. Basics, principles, techniques and modern methods in paediatric

ultrasonography. Eur J Radiol 2014; 83: 1487-1494.

12. Aldrich JE. Basic physics of ultrasound imaging. Crit Care Med 2007; 35: S131-137.

13. Martins WP. Questionable value of absolute mean gray value for clinical practice. Ultrasound

Obstet Gynecol 2013; 41: 595-597.

14. Lima MLS, Nastri CO, Coelho Neto MA, Navarro PA, Martins WP. Antral follicle count might

be underestimated in the presence of an ovarian endometrioma. Hum Reprod 2014; In press.

15. Sanchez AM, Vigano P, Somigliana E, Panina-Bordignon P, Vercellini P, Candiani M. The

distinguishing cellular and molecular features of the endometriotic ovarian cyst: from

pathophysiology to the potential endometrioma-mediated damage to the ovary. Hum Reprod

Update 2014; 20: 217-230.

16. Halis G, Arici A. Endometriosis and inflammation in infertility. Ann N Y Acad Sci 2004; 1034:

300-315.

This article is protected by copyright. All rights reserved

17. Narouze SN. Atlas of ultrasound-guided procedures in interventional pain management. New

York: Springer; 2011:xxviii, 372 p.

18. Alborzi S, Keramati P, Younesi M, Samsami A, Dadras N. The impact of laparoscopic

cystectomy on ovarian reserve in patients with unilateral and bilateral endometriomas. Fertil Steril

2014; 101: 427-434.

19. Kwon SK, Kim SH, Yun SC, Kim DY, Chae HD, Kim CH, Kang BM. Decline of serum

antimullerian hormone levels after laparoscopic ovarian cystectomy in endometrioma and other

benign cysts: a prospective cohort study. Fertil Steril 2014; 101: 435-441.

20. Barbosa MA, Teixeira DM, Navarro PA, Ferriani RA, Nastri CO, Martins WP. Impact of

endometriosis and its staging on assisted reproduction outcome: systematic review and meta-

analysis. Ultrasound Obstet Gynecol 2014; 44: 261-278.

This article is protected by copyright. All rights reserved

Table 1 Comparison of ovarian outcomes between ovaries with and without endometriomas in

women undergoing controlled ovarian stimulation and follicle aspiration for IVF.

Ovary with endometrioma Contralateral ovary

Parametric evaluation Mean ± SD Mean ± SD P*

Ovarian Volume (cm³) 16.5 ± 15.1 4.7 ± 2.7 <0.001

AFC 3.6 ± 3.1 5.1 ± 3.6 0.01

Oocytes retrieved 3.1 ± 3.4 3.0 ± 3.6 0.80

(AFC) – (Oocytes

retrieved)

0.4 ± 3.1 2.1 ± 3.2 0.004

Non parametric

evaluation

Median IQR Range Median IQR Range P**

Ovarian Volume (cm³) 10.3 4.7 to

18.9

2.6 to

57.0

3.6 2.7 to

6.5

0.9 to

10.3

<0.001

AFC 3.0 1.0 to

6.0

0.0 to

10.0

5.0 2.0 to

6.5

0.0 to

14.0

0.001

Oocytes retrieved 2.0 0.5 to

5.0

0.0 to

12.0

2.0 0.0 to

4.0

0.0 to

17.0

0.60

(AFC) – (Oocytes

retrieved)

0.0 -1.0 to

1.5

-6.o to

10.0

2.0 0.0 to

4.0

-5.0 to

12.0

0.005

AFC = antral follicle count; IQR = (Interquartile range); * = p-values determined by paired t tests; ** =

p-values determined by Wilcoxon test.

This article is protected by copyright. All rights reserved

Figure 1 Difference between the antral follicle count (AFC) and the number of oocytes retrieved in

women undergoing controlled ovarian stimulation and follicle aspiration for IVF; comparison

between ovaries with and without endometrioma. The horizontal lines represent the median value.