Hum. Reprod. Advance Access published March 27, 2014 Human Reproduction, Vol.0, No.0 pp. 1 –7, 2014 doi:10.1093/humrep/deu055
ORIGINAL ARTICLE Reproductive genetics
Antral follicle count as a marker of ovarian biological age to reflect the background risk of fetal aneuploidy
1
Department of Maternal-Fetal Medicine, Institute Gynecology, Obstetrics and Neonatology, Hospital Clı´nic Barcelona, Barcelona, Catalonia, Spain 2CIBER de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain 3Department of Gynecology and Reproduction, Institute Gynecology, Obstetrics and Neonatology, Hospital Clı´nic Barcelona, Barcelona, Catalonia, Spain *Correspondence address. Department of Maternal-Fetal Medicine, Institute Gynecology, Obstetrics and Neonatology, Hospital Clı´nic Barcelona, Sabino de Arana 1, Barcelona 08028, Catalonia, Spain. Tel: +34 93 227 99 46; Fax: +34 93 2275605; E-mail: [email protected]
Submitted on November 8, 2013; resubmitted on January 29, 2014; accepted on February 5, 2014
study question: Can antral follicle count (AFC) measured during pregnancy be used as a marker of ovarian age to assess the background risk of fetal aneuploidy?
summary answer: AFC was lower than expected according to maternal chronological age in trisomic pregnancies; therefore ovarian age could potentially reflect a more precise background risk of fetal aneuploidy screening. what is known already: The decline in a woman’s reproductive function is determined by a decline in the ovarian follicle pool and the quality of oocytes. The quantitative status of ovarian reserve can be indirectly assessed by AFC, but the role of AFC as an aneuploidy risk marker in pregnant women has not been assessed yet. study design, size, duration: Our study comprised a prospective cohort including 1239 singleton pregnancies scanned before 14 weeks in our center during a 14-month period.
participants/materials, setting, methods: Reference ranges for AFC were constructed using 812 spontaneously conceived, chromosomally normal singleton ongoing pregnancies using the Lambda-Mu-Sigma method. The study population (n ¼ 934) included 19 pregnancies with viable autosomal trisomies (trisomies 21, 18 and 13), 17 non-viable autosomal trisomies (other than 21, 18 or 13), 7 monosomies X, 1 sex trisomy and 3 triploidies (total n ¼ 47 with chromosomal abnormalities). AFC in chromosomally abnormal pregnancies was plotted against the reference ranges. AFC multiple of the median was calculated according to the median AFC obtained by each year of age. main results and the role of chance: Sixty-eight percent of women carrying a pregnancy with viable trisomies and 65% with non-viable trisomies presented an AFC below the 50th percentile. The median ovarian age in viable trisomies and non-viable trisomies was estimated to be 3 and 6 years above than median maternal age, respectively. However, the median ovarian age in monosomies X and triploidies was not higher than median maternal age.
limitations, reasons for caution: We did not assess the intra- and inter-observer reliability, or use specific three-dimensional analysis which may have advantages over our two-dimensional study. In clinical practice, a drawback for assessing AFC during pregnancy is that transvaginal ultrasound is needed at the 11- to 13-week scan, when the transabdominal approach is used most commonly. Furthermore identifying ovaries by ultrasound during pregnancy could be challenging. wider implications of the findings: Considering that AFC reflects ovarian aging, this ‘ovarian biological age’ could potentially reflect a more precise background risk of fetal aneuploidy. study funding/competing interest(s): This study was supported by PI 11/00685. Instituto de Salud Carlos III. Fondo de Investigacio´n Sanitaria. No competing interests declared. Key words: antral follicle count / ovarian age / chronological age / aneuploid pregnancies / ovarian reserve
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
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Maribel Grande 1,2, Virginia Borobio 1, Jose Miguel Jimenez 1, Mar Bennasar 1, Iosifina Stergiotou 1, Joana Pen˜arrubia 3, and Antoni Borrell 1,2,*
2
Introduction
Materials and Methods Population During a 14-month period (April 2012 – June 2013) pregnant women scanned at 11 –13 weeks in our center were invited to be enrolled in the study. The study population included pregnant women attending for a routine first trimester scan or chorionic villi sampling (CVS), pregnant women with aneuploidy referred from other centers, and women with pregnancy loss up to 14 weeks attending for CVS. Maternal assessment included a medical history and measurement of anthropometric indices. All of the nonkaryotyped pregnancies underwent long-term follow-up. All pregnancies achieved by assisted reproduction techniques (ART) were excluded, as some kind of ovarian stimulation was almost invariably carried out, and oocyte or egg donation may have been involved. Multiple pregnancies were also excluded. Ethics approval was received from the local Research Ethics Committee and written informed consent was obtained from recruited pregnant women.
First trimester scan and AFC First trimester scans were performed by four sonologists using three ultrasound machines (Acuson Antares, Siemens Medical Solutions, Malvern,
PA, USA; Voluson E6 General Electric, GE Healthcare Austria GmbH & Co; and Aloka Prosound a7, Aloka Co. Ltd, Tokyo, Japan). A 25-min slot was assigned to perform both a routine transabdominal first trimester scan and an additional transvaginal scan to assess AFC. Round or oval sonolucent structures in the ovaries, not capturing color flow, were regarded as follicles, but only follicles measuring between 2 and 10 mm mean diameter were included in the AFC. Follicles were grouped as small (2– 5 mm) and large (6– 10 mm). Technical considerations for the assessment of AFC were used, as previously described (Broekmans et al., 2010).
CVS and karyotyping In ongoing pregnancies and in pregnancy losses, CVS was performed transcervically in the vast majority of cases, by means of a round-tip curved steel forceps (1.9 mm diameter and 25 cm long), first introduced by Rodeck (Area Medica, Barcelona, Spain), under continuous ultrasound guidance (Borrell et al., 1999). At the laboratory, samples were inspected under the dissecting microscope to release villi from maternal material. Cytogenetic analysis was carried out after both short- and long-term cultures (Morales et al., 2008; Mun˜oz et al. 2010), while quantitative fluorescent PCR was applied only to ongoing pregnancies (Badenas et al., 2010).
AFC reference ranges Maternal and clinical data were recorded and analyzed in the Statistical Package for the Social Sciences version 17. A P , 0.05 was considered as significant. Normal ranges for AFC were constructed using the Lambda-Mus-Sigma (LMS) method (Cole and Green, 1992) including 812 singleton ongoing pregnancies with normal karyotype not conceived by ART. In brief LMS method summarizes the changing distribution by three curves representing the skewness expressed as a Box-Cox power (L), the median (M) and coefficient of variation (S). The final curves of percentiles are produced by these three smooth curves (Cole and Green, 1992). Degrees of freedom for each curve (L, M and S) were selected according to changes in the model deviance. Centile values were computed by LMS Chartmaker software (LMS Chartmaker Light version 2.54, Medical Research Council, Cambridge, UK). A table reporting the mean and the 90% interval of AFC (5th and 95th percentiles) for each age interval was created.
AFC in chromosomally abnormal pregnancies AFC in mothers of fetuses with viable autosomal trisomies (trisomies 21, 18 and 13), non-viable autosomal trisomies (other than 21, 18 and 13), monosomies X, sex trisomies and triploidies were plotted against the reference ranges constructed here. AFC multiple of the median (MoM) and ovarian age were calculated according to the median AFC obtained by each year of age. Comparison between chromosomally normal and abnormal pregnancy groups was carried out using 95% confidence intervals (95% CI) and the chisquare test. A stratified analysis was carried out taking into account only the small follicles, defined as those with a mean diameter of 2 – 5 mm.
Results A total of 1239 singleton pregnancies were initially included in the study, but 100 cases were excluded because of ART. After ultrasound examination, 205 pregnancies were also excluded due to failure to evaluate antral follicles in one (n ¼ 125; 11%) or both (n ¼ 80; 7%) ovaries. The study population finally included 728 pregnant women attending for routine scans, 101 pregnant women attending for CVS, 8 trisomy 21 pregnancies referred from other centers and 97 women undergoing CVS for pregnancy loss (total n ¼ 934). The mean maternal age was 32.8 years (range: 16 –45 years) and the mean crown-rump length was 60 mm
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Reproductive aging in women is determined by ovarian aging and expressed as a decline in both the quantity of primordial follicles and quality of the oocytes within (Te Velde et al., 1998). Peak fertility occurs in the mid-20s, after which a decline in fertility is observed until menopause (Menken et al., 1986; van Noord-Zaadstra et al., 1991). The wide range of chronological age at which menopause occurs suggests that reproductive aging presents variability between women, and that age alone does not reflect the women’s reproductive potential (El-Toukhy et al., 2002). Although initially follicle decline was described to be biphasic with a sudden acceleration of oocyte decline at 37 years of age (Faddy et al., 1992), subsequent studies suggested a gradual acceleration of oocyte loss over time (Leidy et al., 1998; Hansen et al., 2008). Different tests have been developed to evaluate ovarian reserve, mainly used in Infertility clinics. Whereas endocrine markers, such as anti-Mu¨llerian hormone (AMH), inhibin B, estradiol and FSH, indirectly reflect the early growing follicle population (Broekmans et al., 2007), ultrasound assessment of the antral follicle count (AFC) provides a direct estimation of recruitable follicles (Hendriks et al., 2005). It has been suggested that ovarian rather than chronological age could determine aneuploidy risk during pregnancy, and therefore trisomy risk would increase with a declining oocyte pool. Thus, it was described that the earlier cessation of reproductive life, brought on by unilateral oophorectomy in female mice, resulted in earlier onset of irregular cyclicity and an earlier rise in aneuploidy (Brook et al., 1984). Moreover, it was demonstrated that menopause occurs at an earlier age among women with trisomic pregnancies (Kline et al., 2000) and that women after a Down syndrome birth present higher values of FSH when compared with controls (van Montfrans et al., 1999). On the above evidence, we aimed to construct reference values for AFC in pregnant women according to maternal age, and subsequently to assess the role of AFC as an ovarian age marker in aneuploid pregnancies. Although there is a small series reporting the AFC in women with proven fertility (Kline et al., 2004), to our knowledge, this is the first study assessing AFC in a cohort of pregnant women.
Grande et al.
3
Antral follicle count in aneuploid pregnancies
Table I Maternal characteristics of women included in the study of antral follicle count (AFC) and risk of fetal aneuploidy. Chromosomally normal pregnancies
Chromosomally abnormal pregnancies
P-value
........................................................................................ Maternal age (years, mean)
32.7
35.5
0.001
BMI (kg/m2, mean)
24.1
23.1
0.751
40.6
Previous live births(%)
38.3
51.1 42.2
0.165 0.602
(range: 3–89 mm). Maternal characteristics from women included in the study are displayed in Table I. Reference ranges were constructed using 812 spontaneously conceived, chromosomally normal singleton ongoing pregnancies. The 5th, 50th and 95th percentiles according to maternal age are presented in Table II. As maternal age rose from 16 to 45 years, the 50th percentile for AFC declined from 23 to 6, whereas the 5th percentile declined from 12 to 2 and the 95th percentile from 41 to 12. Among the observed 47 pregnancies with chromosomal abnormalities, 17 were found after CVS was performed in ongoing pregnancies, 7 pregnancies were referrals and 23 cases of aneuploidy were detected in pregnancy losses. Chromosomal anomaly categories were classified as follows: 19 viable autosomal trisomies, 17 non-viable autosomal trisomies, 7 monosomies X, 1 sex trisomy and 3 triploidies. In Fig. 1, the AFC observed in women with chromosomal anomalies of the fetus were plotted on the age-specific reference ranges for each category. Observed AFC and derived AFC MoM for each of the 47 chromosomal anomalies are displayed in Table III. The frequency of women with AFC below the 50th percentile was 68% (13/19, 95% CI 48–89%, P ¼ 0.11) for viable trisomies, 65% (11/17, 95% CI 42–87%, P ¼ 0.23) for non-viable trisomies, 43% (3/7, 95% CI 6–80%, P ¼ 0.71) for monosomies X, 100% (1/1, 95% CI 100–100%, P ¼ 0.32) for sex trisomies and 33% (1/3, 95% CI 0–86%, P ¼ 0.56) for triploidies. Maternal age and derived ovarian age for mothers of each of the 47 chromosomally abnormal pregnancies are also displayed in Table II. The median ovarian age in viable trisomies and non-viable trisomies was estimated to be 3 and 6 years higher than the median maternal age, respectively. However, the median ovarian age in monosomies X and triploidies was not higher than median maternal age. When the ovarian age is recalculated taking into account only the small antral follicles (2– 5 mm), the resulting median ovarian age, in the different chromosomal anomalies, is about 1 year lower than that obtained with the total AFC (41 years for viable trisomies, 40 for non-viable trisomies, 29.5 for sex monosomies, 37 for sex trisomies and 29.5 for triploidies), and therefore diverged less from the maternal chronological age.
Discussion In our study, for the first time, AFC reference ranges were constructed in a cohort of pregnant women. Our results provide evidence that the
Maternal age (years)
N
AFC (n)
.......................................................... 5th percentile
50th percentile
95th percentile
........................................................................................ 16
1
12
23
41
17
0
11
22
41
18
5
11
22
40
19
9
11
21
39
20
9
10
21
39
21
8
10
20
38
22
7
10
20
38
23
10
10
19
38
24
18
9
19
37
25
24
9
19
37
26
19
9
18
36
27
17
8
18
36
28
30
8
17
35
29
42
8
17
35
30
52
7
16
34
31
62
7
16
33
32
63
7
15
32
33
66
6
15
31
34
68
6
15
30
35
63
6
14
29
36
56
5
14
28
37
38
5
13
27
38
48
5
13
26
39
34
4
12
24
40
30
4
11
22
41
17
3
10
20
42
10
3
9
18
43
3
2
8
16
44
1
2
7
14
45
2
2
6
12
ovarian biological age in trisomic pregnancies, but not in pregnancies with monosomy X or triploidy, is higher than chronological maternal age, suggesting a lower ovarian reserve. Several hormonal and ultrasound markers of ovarian reserve have been explored to identify women with a poor response to ovulation induction in ART. The more recently identified markers, AMH and AFC have found to be more useful than markers previously described such as early follicular FSH and early follicular phase inhibin B (Toner et al., 1991; Seifer et al., 1999). Serum AMH level indirectly reflects the size of the remaining primordial follicle pool and has been shown to have limited inter- and intra-cycle variability (La Marca et al., 2007). The ultrasound assessment of AFC in the early follicular phase directly correlates with ovarian reserve (Frattarelli et al., 2000) and its decline could be a sign
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Previous losses (%)
Table II AFC reference ranges according to woman’s age as observed in 812 pregnant women with a spontaneously conceived chromosomally normal singleton fetus.
4
Grande et al.
ranges (5th, 50th and 95th percentiles).
of ovarian aging (Scheffer et al., 1999). Our study was the first to assess the AFC in a cohort of pregnant woman, since previous studies were mainly conducted in infertility patients (Chang et al., 1998; Jayaprakasan et al., 2010; Muttukrishna et al., 2005) or by histological analysis, after oophorectomy surgery and in post-portem studies (Gougeon et al., 1994; Hansen et al., 2008). Our study, one of the largest on AFC, supports a gradual AFC decline with chronological age. This finding is in agreement with the prior studies (Leidy et al., 1998; Hansen et al., 2008), although our results indicated a higher starting AFC and steeper decline. Namely, in the age range of 22 – 45 years, our series showed that the 50th centile declined from 23 to 6 follicles, while in the La Marca et al. (2011) series the observed declined was from 14 to 5 follicles, and in the Wiweko et al. (2013) series from 9 to 5 follicles. The decline for the 5th centile was from 12 to 2 follicles in our study, from 5 to 2 (La Marca et al., 2011) and from 4 to 1 follicles (Wiweko et al., 2013). However, comparisons with the current study should be made with caution as the study populations differed. Our study comprised pregnant women with spontaneous conception, while Wiweko et al. (2013) included infertile patients undergoing IVF, and La Marca et al. (2011) recruited healthy women with regular menstrual cycles attending gynaecological clinics in order to achieve a sample close to the general population. Only a small group of 65 parous women studied by Kline et al. (2004), used as control group, had a similar AFC to our pregnant population, given that the regression line decline went from 23 follicles at 22 years to 8 follicles at 42 years of age. Although the role of AFC assessment in ART is well established, scarce data are available on its association with trisomic pregnancies and its use in aneuploidy prenatal screening programs is uncertain. Our results are in
line with previous studies linking low ovarian reserve, and therefore poor oocyte quantity and quality, with trisomic offspring risk at any maternal chronological age. Thus, trisomy risk would increase with a decreasing oocyte pool size as a result of an accelerated rate of oocyte atresia (Brook et al., 1984; Kline et al., 2000). This hypothesis suggests that with a decreasing oocyte count, oocyte selection becomes impaired and the hormonal environment of the remaining oocytes may harm their integrity and quality (Warburton, 1989; Dursun et al., 2006; McTavish et al., 2007). In our study, AFC was lower than expected based on reference values in viable (trisomies 21, 18 and 13) and non-viable (other) trisomies, but not in monosomies X or triploidies, which is in accordance with the described association between distinct chromosomal anomalies and advanced maternal age. Accordingly, MoM AFC values presented a 15% reduction (0.85 MoM) in women with viable and nonviable trisomic pregnancies, while in monosomy X or triploidy normal values were observed. Subsequently, the AFC reference ranges constructed in our study allowed us to assign an ‘ovarian biological age’ to each of the pregnant women with a chromosomal abnormality. The resulting ‘ovarian biological age’ was higher than chronological age in trisomic pregnancies, as suggested by several previous studies. Firstly, women with a trisomy 21 child have been reported to present more often with a history of ovarian surgery or absence of an ovary (Freeman et al., 2000), reproducing experimental studies in mice after unilateral oophorectomy (Brook et al., 1984). Secondly, the beginning of menopause was observed to occur 1 year earlier among women with trisomic pregnancy losses (Kline et al., 2000). Thirdly, a low oocyte yield (≤4 oocytes) in women undergoing IVF was found to be associated with an increased risk of a trisomic pregnancy (Haadsma et al., 2010). Conversely, some studies have not found differences in
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Figure 1 Antral follicle count (AFC) observed in pregnant women carrying a singleton chromosomally abnormal fetus plotted against the reference
5
Antral follicle count in aneuploid pregnancies
Table III Individual maternal chronological age, AFC and AFC multiple of the median (MoM) values observed in women carrying a singleton chromosomally abnormal fetus, together with the corresponding ovarian biological age derived from the reference curves. Karyotype
Maternal age (years)
AFC (n)
AFC (MoM)
Ovarian age (years)
............................................................................................................................................................................................. Viable trisomies
Monosomies X
40
4
0.36
.45
39
8
0.66
43
47,XX+21
33
9
0.6
42
47,XX+21
34
14
0.93
36
47,XX+21
41
16
1.6
31
47,XX+21
38
19
1.46
25
47,XY+21
29
28
1.64
,16
47,XY+21
36
32
2.28
,16
47,XX+21
40
5
0.45
.45
47,XX+21
44
6
0.85
45
47,XX+21
41
9
0.9
42
47,XY+21
42
1
0.11
47,XY+21
42
9
47,XY+21
36
10
47,XX+18
44
9
1.28
42
47,XX+18
33
12
0.8
39
47,XY,+18
39
9
0.75
42
47,XY,+18
39
10
0.83
41
47,XY+13
36
12
0.85
39
Median
39
9
0.85
42 45
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Non-viable trisomies
47,XY+21 47,XX+21
.45
1
42
0.71
41
47,XY+9
39
6
0.5
47,XY+8
42
8
0.88
43
47,XY,+7
39
10
0.83
41
47,XY,+i(12)(p10)
34
7
0.46
44
47,XY+15
27
10
0.55
41
47,XY,+15
42
1
0.11
.45
47,XY,+16
34
7
0.46
44
47,XY,+16
31
21
1.31
20
47,XY+16
34
14
0.93
36
47,XY+16
33
16
1.06
31
47,XX,+16
31
18
1.12
27
47,XY+20
36
7
0.5
44
47,XX,+20
37
5
0.38
.45
47,XY,+22
33
13
0.86
38
47,XY,+22
38
20
1.53
22
47,XY+22
36
23
1.64
16
47,X,+mar,+mar
35
17
1.21
29
Median
35
10
0.86
41
45,X
30
7
0.43
44
45,X
24
15
0.78
34
45,X
40
16
1.45
31
45,X
26
18
1
45,X
37
25
1.92
,16
45,X
27
31
1.72
,16
27
mos 45,X[26]/46,XY[24]
38
8
0.61
43
Median
30
16
1.00
30.5
Continued
6
Grande et al.
Table III Continued Karyotype
Maternal age (years)
AFC (n)
AFC (MoM)
Ovarian age (years)
............................................................................................................................................................................................. Sex trisomies
47XYY
26
12
0.66
39
Triploidies
69,XXX
30
14
0.87
36
69,XXX
32
16
1.06
31
69,XXY
32
25
1.66
,16
Median
32
16
1.06
association and the potential utility of our measurements to identify mothers at higher risk of trisomic pregnancies.
Acknowledgements The authors thank Cristina Cobos, Sonia Matias and Narcis Masoller for helping in the acquisition of samples.
Authors’ roles M.G. and A.B. carried out the study design, the analysis and interpretation of data and the draft of the manuscript. A.B., V.B., J.M.J., I.S. and M.B. contributed in the acquisition of samples and revised the draft. A.B. and J.P. revised the article for important intellectual content.
Funding This study was supported by PI 11/00685. Instituto de Salud Carlos III. Fondo de Investigacio´n Sanitaria.
Conflict of interest None declared.
References Badenas C, Rodrı´guez-Revenga L, Morales C, Mediano C, Plaja A, Pe´rez-Iribarne MM, Soler A, Clusellas N, Borrell A, Sa´nchez MA´ et al. Assessment of QF-PCR as the first approach in prenatal diagnosis. J Mol Diagn 2010;12:828 – 834. Benn P, Borrell A, Cuckle H, Dugoff L, Gross S, Johnson JA, Maymon R, Odibo A, Schielen P, Spencer K et al. Prenatal Detection of Down Syndrome using Massively Parallel Sequencing (MPS): a rapid response statement from a committee on behalf of the Board of the International Society for Prenatal Diagnosis, 24 October 2011. Prenat Diagn 2012;32:1–2. Borrell A, Fortuny A, Lazaro L, Costa D, Seres A, Pappa S, Soler A. First-trimester transcervical chorionic villus sampling by biopsy forceps versus mid-trimester amniocentesis: a randomized controlled trial project. Prenat Diagn 1999;19:1138 – 1142. Borrell A, Casals E, Fortuny A, Farre MT, Gonce A, Sanchez A, Soler A, Cararach V, Vanrell JA. First-trimester screening for trisomy 21 combining biochemistry and ultrasound at individually optimal gestational ages: an interventional study. Prenat Diagn 2004;24:541 – 545. Broekmans FJ, Knauff EA, te Velde ER, Macklon NS, Fauser BC. Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metab 2007;18:58 – 65.
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AFC, inhibin B or FSH between trisomic and control pregnancies (Kline et al., 2004). The Warburton group found in a recent study differences only in FSH (Kline et al., 2011), not supporting the oocyte pool hypothesis, given that AMH was more strongly associated with chronologic age than FSH (de Vet et al., 2002). Assessing the ovarian reserve during pregnancy may have important clinical implications. Considering that AFC reflects ovarian aging, this ‘ovarian biological age’ may be a better aneuploidy risk marker than maternal age. The relationship between advanced maternal age and increased risk of fetal aneuploidy was established many decades ago (Cuckle et al., 1987). Down syndrome risk was initially estimated according exclusively to maternal age, while later this was taken as the background risk to be modified by serum maternal markers (Wald et al., 1988). Currently, cell-free fetal DNA analysis in maternal blood can also be clinically applied as a likelihood ratio to modify this background risk (Benn et al., 2012). Thus, although the use of maternal age as an exclusive criterion for increased risk of fetal aneuploidy is being abandoned worldwide, most of the Down syndrome screening models are based on refining the maternal age background risk. The strength of our study was mainly to construct the reference ranges from a large study population of 812 women, more than double the number of women compared with the previous largest series of 366 women (Wiweko et al., 2013). Furthermore, our series is the first to assess the AFC in pregnant women, demonstrating that this is feasible during the routine 11- to 13-week scan at the moment of aneuploidy risk assessment (Borrell et al., 2004). Although most of the AFC studies were focused on predicting the ovarian yield in infertile couples, our outcome measure was to determine the ‘ovarian biological age’ in women with regular cycles and spontaneous conception. We acknowledge some limitations of our study due to not performing intra- and inter-observer reliability analysis, and not using specific 3D software (SonoAVC), shown by some authors to be the best method for assessing AFC if postprocessing correction is carried out (Deb et al., 2009). Instead, we adhered to the Broekmans guidelines for 2D assessment, a well-established standardized approach to reduce AFC variability (Broekmans et al., 2010). In clinical practice, a drawback for assessing AFC is that transvaginal ultrasound is needed during the 11- to 13-week scan, when the transabdominal approach is the most commonly used. Furthermore, identifying ovaries by ultrasound during pregnancy could be challenging, particularly when the time slot assigned is limited. However, with appropriate training and motivation, sonologists can achieve this skill. In summary, current data suggest that ovarian biological age could potentially reflect a more precise background risk of fetal aneuploidy. Future studies are warranted to assess the involved complex pathophysiologic
30.5
Antral follicle count in aneuploid pregnancies
Kline J, Kinney A, Reuss ML, Kelly A, Levin B, Ferin M, Warburton D. Trisomic pregnancy and the oocyte pool. Hum Reprod 2004;19:1633– 1643. Kline JK, Kinney AM, Levin B, Kelly AC, Ferin M, Warburton D. Trisomic pregnancy and elevated FSH: implications for the oocyte pool hypothesis. Hum Reprod 2011;26:1537– 1550. La Marca A, Giulini S, Tirelli A, Bertucci E, Marsella T, Xella S, Volpe A. Anti-Mu¨llerian hormone measurement on any day of the menstrual cycle strongly predicts ovarian response in assisted reproductive technology. Hum Reprod 2007;22:766 – 771. La Marca A, Spada E, Sighinolfi G, Argento C, Tirelli A, Giulini S, Milani S, Volpe A. Age-specific nomogram for the decline in antral follicle count throughout the reproductive period. Fertil Steril 2011;95:684 – 688. Leidy LE, Godfrey LR, Sutherland MR. Is follicular atresia biphasic? Fertil Steril 1998;70:851 – 859. McTavish KJ, Jimenez M, Walters KA, Spaliviero J, Groome NP, Themmen AP, Visser JA, Handelsman DJ, Allan CM. Rising follicle-stimulating hormone levels with age accelerate female reproductive failure. Endocrinology 2007; 148:4432–4439. Menken J, Trussell J, Larsen U. Age and infertility. Science 1986; 233:1389– 1394. Morales C, Sa´nchez A, Bruguera J, Margarit E, Borrell A, Borobio V, Soler A. Cytogenetic study of spontaneous abortions using semi-direct analysis of chorionic villi samples detects the broadest spectrum of chromosome abnormalities. Am J Med Genet A 2008;146A:66 – 70. Mun˜oz M, Arigita M, Bennasar M, Soler A, Sanchez A, Borrell A. Chromosomal anomaly spectrum in early pregnancy loss in relation to presence or absence of an embryonic pole. Fertil Steril 2010; 94:2564– 2568. Muttukrishna S, McGarrigle H, Wakim R, Khadum I, Ranieri DM, Serhal P. Antral follicle count, anti-mullerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology? BJOG 2005; 112:1384– 1390. Scheffer GJ, Broekmans FJ, Dorland M, Habbema JD, Looman CW, te Velde ER. Antral follicle counts by transvaginal ultrasonography are related to age in women with proven natural fertility. Fertil Steril 1999; 72:845– 851. Seifer DB, Scott RT Jr, Bergh PA, Abrogast LK, Friedman CI, Mack CK, Danforth DR. Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone. Fertil Steril 1999;72:63 – 65. te Velde ER, Scheffer GJ, Dorland M, Broekmans FJ, Fauser BC. Developmental and endocrine aspects of normal ovarian aging. Mol Cell Endocrinol 1998;145:67 – 73. Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril 1991;55:784 – 791. van Montfrans JM, Dorland M, Oosterhuis GJ, van Vugt JM, Rekers-Mombarg LT, Lambalk CB. Increased concentrations of follicle-stimulating hormone in mothers of children with Down’s syndrome. Lancet 1999;353:1853– 1854. van Noord-Zaadstra BM, Looman CW, Alsbach H, Habbema JD, te Velde ER, Karbaat J. Delaying childbearing: effect of age on fecundity and outcome of pregnancy. BMJ 1991;302:1361 – 1365. Wald NJ, Cuckle HS, Densem JW, Nanchahal K, Royston P, Chard T, Haddow JE, Knight GJ, Palomaki GE, Canick JA. Maternal serum screening for Down’s syndrome in early pregnancy. BMJ 1988;297:883–887. Warburton D. The effect of maternal age on the frequency of trisomy: change in meiosis or in utero selection? Prog Clin Biol Res 1989;311:165 – 181. Wiweko B, Prawesti DM, Hestiantoro A, Sumapraja K, Natadisastra M, Baziad A. Chronological age vs biological age: an age-related normogram for antral follicle count, FSH and anti-Mullerian hormone. J Assist Reprod Genet. 2013;30:1563– 1567.
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Broekmans FJ, de Ziegler D, Howles CM, Gougeon A, Trew G, Olivennes F. The antral follicle count: practical recommendations for better standardization. Fertil Steril 2010;94:1044 – 1051. Brook JD, Gosden RG, Chandley AC. Maternal ageing and aneuploid embryos—evidence from the mouse that biological and not chronological age is the important influence. Hum Genet 1984;66: 41 – 45. Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. Use of the antral follicle count to predict the outcome of assisted reproductive technologies. Fertil Steril 1998;69:505 – 510. Cole TJ, Green PJ. Smoothing reference centile curves: the LMS method and penalized likelihood. Stat Med 1992;11:1305 –1319. Cuckle HS, Wald NJ, Thompson SG. Estimating a woman’s risk of having a pregnancy associated with Down’s syndrome using her age and serum alpha-fetoprotein level. Br J Obstet Gynaecol 1987;94:387 – 402. Deb S, Jayaprakasan K, Campbell BK, Clewes JS, Johnson IR, Raine-Fenning NJ. Intraobserver and interobserver reliability of automated antral follicle counts made using three-dimensional ultrasound and SonoAVC. Ultrasound Obstet Gynecol 2009;33:477 – 483. de Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC. Antimu¨llerian hormone serum levels: a putative marker for ovarian aging. Fertil Steril 2002;77:357 – 362. Dursun P, Gultekin M, Yuce K, Ayhan A. What is the underlying cause of aneuploidy associated with increasing maternal age? Is it associated with elevated levels of gonadotropins? Med Hypotheses 2006;66:143– 147. El-Toukhy T, Khalaf Y, Hart R, Taylor A, Braude P. Young age does not protect against the adverse effects of reduced ovarian reserve—an eight year study. Hum Reprod 2002;17:1519– 1524. Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342– 1346. Frattarelli JL, Lauria-Costab DF, Miller BT, Bergh PA, Scott RT. Basal antral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles. Fertil Steril 2000;74:512 –517. Freeman SB, Yang Q, Allran K, Taft LF, Sherman SL. Women with a reduced ovarian complement may have an increased risk for a child with Down syndrome. Am J Hum Genet 2000;66:1680 – 1683. Gougeon A, Ecochard R, Thalabard JC. Age-related changes of the population of human ovarian follicles: increase in the disappearance rate of non-growing and early-growing follicles in aging women. Biol Reprod 1994;50:653 – 663. Haadsma ML, Mooij TM, Groen H, Burger CW, Lambalk CB, Broekmans FJ, van Leeuwen FE, Bouman K, Hoek A. OMEGA Project Group. A reduced size of the ovarian follicle pool is associated with an increased risk of a trisomic pregnancy in IVF-treated women. Hum Reprod 2010; 25:552– 558. Hansen KR, Knowlton NS, Thyer AC, Charleston JS, Soules MR, Klein NA. A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause. Hum Reprod 2008;23: 699 – 708. 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. Jayaprakasan K, Deb S, Batcha M, Hopkisson J, Johnson I, Campbell B, Raine-Fenning N. The cohort of antral follicles measuring 2 –6 mm reflects the quantitative status of ovarian reserve as assessed by serum levels of anti-Mu¨llerian hormone and response to controlled ovarian stimulation. Fertil Steril 2010;94:1775 – 1781. Kline J, Kinney A, Levin B, Warburton D. Trisomic pregnancy and earlier age at menopause. Am J Hum Genet 2000;67:395 – 404.
7
ORIGINAL ARTICLE Reproductive genetics
Antral follicle count as a marker of ovarian biological age to reflect the background risk of fetal aneuploidy
1
Department of Maternal-Fetal Medicine, Institute Gynecology, Obstetrics and Neonatology, Hospital Clı´nic Barcelona, Barcelona, Catalonia, Spain 2CIBER de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain 3Department of Gynecology and Reproduction, Institute Gynecology, Obstetrics and Neonatology, Hospital Clı´nic Barcelona, Barcelona, Catalonia, Spain *Correspondence address. Department of Maternal-Fetal Medicine, Institute Gynecology, Obstetrics and Neonatology, Hospital Clı´nic Barcelona, Sabino de Arana 1, Barcelona 08028, Catalonia, Spain. Tel: +34 93 227 99 46; Fax: +34 93 2275605; E-mail: [email protected]
Submitted on November 8, 2013; resubmitted on January 29, 2014; accepted on February 5, 2014
study question: Can antral follicle count (AFC) measured during pregnancy be used as a marker of ovarian age to assess the background risk of fetal aneuploidy?
summary answer: AFC was lower than expected according to maternal chronological age in trisomic pregnancies; therefore ovarian age could potentially reflect a more precise background risk of fetal aneuploidy screening. what is known already: The decline in a woman’s reproductive function is determined by a decline in the ovarian follicle pool and the quality of oocytes. The quantitative status of ovarian reserve can be indirectly assessed by AFC, but the role of AFC as an aneuploidy risk marker in pregnant women has not been assessed yet. study design, size, duration: Our study comprised a prospective cohort including 1239 singleton pregnancies scanned before 14 weeks in our center during a 14-month period.
participants/materials, setting, methods: Reference ranges for AFC were constructed using 812 spontaneously conceived, chromosomally normal singleton ongoing pregnancies using the Lambda-Mu-Sigma method. The study population (n ¼ 934) included 19 pregnancies with viable autosomal trisomies (trisomies 21, 18 and 13), 17 non-viable autosomal trisomies (other than 21, 18 or 13), 7 monosomies X, 1 sex trisomy and 3 triploidies (total n ¼ 47 with chromosomal abnormalities). AFC in chromosomally abnormal pregnancies was plotted against the reference ranges. AFC multiple of the median was calculated according to the median AFC obtained by each year of age. main results and the role of chance: Sixty-eight percent of women carrying a pregnancy with viable trisomies and 65% with non-viable trisomies presented an AFC below the 50th percentile. The median ovarian age in viable trisomies and non-viable trisomies was estimated to be 3 and 6 years above than median maternal age, respectively. However, the median ovarian age in monosomies X and triploidies was not higher than median maternal age.
limitations, reasons for caution: We did not assess the intra- and inter-observer reliability, or use specific three-dimensional analysis which may have advantages over our two-dimensional study. In clinical practice, a drawback for assessing AFC during pregnancy is that transvaginal ultrasound is needed at the 11- to 13-week scan, when the transabdominal approach is used most commonly. Furthermore identifying ovaries by ultrasound during pregnancy could be challenging. wider implications of the findings: Considering that AFC reflects ovarian aging, this ‘ovarian biological age’ could potentially reflect a more precise background risk of fetal aneuploidy. study funding/competing interest(s): This study was supported by PI 11/00685. Instituto de Salud Carlos III. Fondo de Investigacio´n Sanitaria. No competing interests declared. Key words: antral follicle count / ovarian age / chronological age / aneuploid pregnancies / ovarian reserve
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
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Maribel Grande 1,2, Virginia Borobio 1, Jose Miguel Jimenez 1, Mar Bennasar 1, Iosifina Stergiotou 1, Joana Pen˜arrubia 3, and Antoni Borrell 1,2,*
2
Introduction
Materials and Methods Population During a 14-month period (April 2012 – June 2013) pregnant women scanned at 11 –13 weeks in our center were invited to be enrolled in the study. The study population included pregnant women attending for a routine first trimester scan or chorionic villi sampling (CVS), pregnant women with aneuploidy referred from other centers, and women with pregnancy loss up to 14 weeks attending for CVS. Maternal assessment included a medical history and measurement of anthropometric indices. All of the nonkaryotyped pregnancies underwent long-term follow-up. All pregnancies achieved by assisted reproduction techniques (ART) were excluded, as some kind of ovarian stimulation was almost invariably carried out, and oocyte or egg donation may have been involved. Multiple pregnancies were also excluded. Ethics approval was received from the local Research Ethics Committee and written informed consent was obtained from recruited pregnant women.
First trimester scan and AFC First trimester scans were performed by four sonologists using three ultrasound machines (Acuson Antares, Siemens Medical Solutions, Malvern,
PA, USA; Voluson E6 General Electric, GE Healthcare Austria GmbH & Co; and Aloka Prosound a7, Aloka Co. Ltd, Tokyo, Japan). A 25-min slot was assigned to perform both a routine transabdominal first trimester scan and an additional transvaginal scan to assess AFC. Round or oval sonolucent structures in the ovaries, not capturing color flow, were regarded as follicles, but only follicles measuring between 2 and 10 mm mean diameter were included in the AFC. Follicles were grouped as small (2– 5 mm) and large (6– 10 mm). Technical considerations for the assessment of AFC were used, as previously described (Broekmans et al., 2010).
CVS and karyotyping In ongoing pregnancies and in pregnancy losses, CVS was performed transcervically in the vast majority of cases, by means of a round-tip curved steel forceps (1.9 mm diameter and 25 cm long), first introduced by Rodeck (Area Medica, Barcelona, Spain), under continuous ultrasound guidance (Borrell et al., 1999). At the laboratory, samples were inspected under the dissecting microscope to release villi from maternal material. Cytogenetic analysis was carried out after both short- and long-term cultures (Morales et al., 2008; Mun˜oz et al. 2010), while quantitative fluorescent PCR was applied only to ongoing pregnancies (Badenas et al., 2010).
AFC reference ranges Maternal and clinical data were recorded and analyzed in the Statistical Package for the Social Sciences version 17. A P , 0.05 was considered as significant. Normal ranges for AFC were constructed using the Lambda-Mus-Sigma (LMS) method (Cole and Green, 1992) including 812 singleton ongoing pregnancies with normal karyotype not conceived by ART. In brief LMS method summarizes the changing distribution by three curves representing the skewness expressed as a Box-Cox power (L), the median (M) and coefficient of variation (S). The final curves of percentiles are produced by these three smooth curves (Cole and Green, 1992). Degrees of freedom for each curve (L, M and S) were selected according to changes in the model deviance. Centile values were computed by LMS Chartmaker software (LMS Chartmaker Light version 2.54, Medical Research Council, Cambridge, UK). A table reporting the mean and the 90% interval of AFC (5th and 95th percentiles) for each age interval was created.
AFC in chromosomally abnormal pregnancies AFC in mothers of fetuses with viable autosomal trisomies (trisomies 21, 18 and 13), non-viable autosomal trisomies (other than 21, 18 and 13), monosomies X, sex trisomies and triploidies were plotted against the reference ranges constructed here. AFC multiple of the median (MoM) and ovarian age were calculated according to the median AFC obtained by each year of age. Comparison between chromosomally normal and abnormal pregnancy groups was carried out using 95% confidence intervals (95% CI) and the chisquare test. A stratified analysis was carried out taking into account only the small follicles, defined as those with a mean diameter of 2 – 5 mm.
Results A total of 1239 singleton pregnancies were initially included in the study, but 100 cases were excluded because of ART. After ultrasound examination, 205 pregnancies were also excluded due to failure to evaluate antral follicles in one (n ¼ 125; 11%) or both (n ¼ 80; 7%) ovaries. The study population finally included 728 pregnant women attending for routine scans, 101 pregnant women attending for CVS, 8 trisomy 21 pregnancies referred from other centers and 97 women undergoing CVS for pregnancy loss (total n ¼ 934). The mean maternal age was 32.8 years (range: 16 –45 years) and the mean crown-rump length was 60 mm
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Reproductive aging in women is determined by ovarian aging and expressed as a decline in both the quantity of primordial follicles and quality of the oocytes within (Te Velde et al., 1998). Peak fertility occurs in the mid-20s, after which a decline in fertility is observed until menopause (Menken et al., 1986; van Noord-Zaadstra et al., 1991). The wide range of chronological age at which menopause occurs suggests that reproductive aging presents variability between women, and that age alone does not reflect the women’s reproductive potential (El-Toukhy et al., 2002). Although initially follicle decline was described to be biphasic with a sudden acceleration of oocyte decline at 37 years of age (Faddy et al., 1992), subsequent studies suggested a gradual acceleration of oocyte loss over time (Leidy et al., 1998; Hansen et al., 2008). Different tests have been developed to evaluate ovarian reserve, mainly used in Infertility clinics. Whereas endocrine markers, such as anti-Mu¨llerian hormone (AMH), inhibin B, estradiol and FSH, indirectly reflect the early growing follicle population (Broekmans et al., 2007), ultrasound assessment of the antral follicle count (AFC) provides a direct estimation of recruitable follicles (Hendriks et al., 2005). It has been suggested that ovarian rather than chronological age could determine aneuploidy risk during pregnancy, and therefore trisomy risk would increase with a declining oocyte pool. Thus, it was described that the earlier cessation of reproductive life, brought on by unilateral oophorectomy in female mice, resulted in earlier onset of irregular cyclicity and an earlier rise in aneuploidy (Brook et al., 1984). Moreover, it was demonstrated that menopause occurs at an earlier age among women with trisomic pregnancies (Kline et al., 2000) and that women after a Down syndrome birth present higher values of FSH when compared with controls (van Montfrans et al., 1999). On the above evidence, we aimed to construct reference values for AFC in pregnant women according to maternal age, and subsequently to assess the role of AFC as an ovarian age marker in aneuploid pregnancies. Although there is a small series reporting the AFC in women with proven fertility (Kline et al., 2004), to our knowledge, this is the first study assessing AFC in a cohort of pregnant women.
Grande et al.
3
Antral follicle count in aneuploid pregnancies
Table I Maternal characteristics of women included in the study of antral follicle count (AFC) and risk of fetal aneuploidy. Chromosomally normal pregnancies
Chromosomally abnormal pregnancies
P-value
........................................................................................ Maternal age (years, mean)
32.7
35.5
0.001
BMI (kg/m2, mean)
24.1
23.1
0.751
40.6
Previous live births(%)
38.3
51.1 42.2
0.165 0.602
(range: 3–89 mm). Maternal characteristics from women included in the study are displayed in Table I. Reference ranges were constructed using 812 spontaneously conceived, chromosomally normal singleton ongoing pregnancies. The 5th, 50th and 95th percentiles according to maternal age are presented in Table II. As maternal age rose from 16 to 45 years, the 50th percentile for AFC declined from 23 to 6, whereas the 5th percentile declined from 12 to 2 and the 95th percentile from 41 to 12. Among the observed 47 pregnancies with chromosomal abnormalities, 17 were found after CVS was performed in ongoing pregnancies, 7 pregnancies were referrals and 23 cases of aneuploidy were detected in pregnancy losses. Chromosomal anomaly categories were classified as follows: 19 viable autosomal trisomies, 17 non-viable autosomal trisomies, 7 monosomies X, 1 sex trisomy and 3 triploidies. In Fig. 1, the AFC observed in women with chromosomal anomalies of the fetus were plotted on the age-specific reference ranges for each category. Observed AFC and derived AFC MoM for each of the 47 chromosomal anomalies are displayed in Table III. The frequency of women with AFC below the 50th percentile was 68% (13/19, 95% CI 48–89%, P ¼ 0.11) for viable trisomies, 65% (11/17, 95% CI 42–87%, P ¼ 0.23) for non-viable trisomies, 43% (3/7, 95% CI 6–80%, P ¼ 0.71) for monosomies X, 100% (1/1, 95% CI 100–100%, P ¼ 0.32) for sex trisomies and 33% (1/3, 95% CI 0–86%, P ¼ 0.56) for triploidies. Maternal age and derived ovarian age for mothers of each of the 47 chromosomally abnormal pregnancies are also displayed in Table II. The median ovarian age in viable trisomies and non-viable trisomies was estimated to be 3 and 6 years higher than the median maternal age, respectively. However, the median ovarian age in monosomies X and triploidies was not higher than median maternal age. When the ovarian age is recalculated taking into account only the small antral follicles (2– 5 mm), the resulting median ovarian age, in the different chromosomal anomalies, is about 1 year lower than that obtained with the total AFC (41 years for viable trisomies, 40 for non-viable trisomies, 29.5 for sex monosomies, 37 for sex trisomies and 29.5 for triploidies), and therefore diverged less from the maternal chronological age.
Discussion In our study, for the first time, AFC reference ranges were constructed in a cohort of pregnant women. Our results provide evidence that the
Maternal age (years)
N
AFC (n)
.......................................................... 5th percentile
50th percentile
95th percentile
........................................................................................ 16
1
12
23
41
17
0
11
22
41
18
5
11
22
40
19
9
11
21
39
20
9
10
21
39
21
8
10
20
38
22
7
10
20
38
23
10
10
19
38
24
18
9
19
37
25
24
9
19
37
26
19
9
18
36
27
17
8
18
36
28
30
8
17
35
29
42
8
17
35
30
52
7
16
34
31
62
7
16
33
32
63
7
15
32
33
66
6
15
31
34
68
6
15
30
35
63
6
14
29
36
56
5
14
28
37
38
5
13
27
38
48
5
13
26
39
34
4
12
24
40
30
4
11
22
41
17
3
10
20
42
10
3
9
18
43
3
2
8
16
44
1
2
7
14
45
2
2
6
12
ovarian biological age in trisomic pregnancies, but not in pregnancies with monosomy X or triploidy, is higher than chronological maternal age, suggesting a lower ovarian reserve. Several hormonal and ultrasound markers of ovarian reserve have been explored to identify women with a poor response to ovulation induction in ART. The more recently identified markers, AMH and AFC have found to be more useful than markers previously described such as early follicular FSH and early follicular phase inhibin B (Toner et al., 1991; Seifer et al., 1999). Serum AMH level indirectly reflects the size of the remaining primordial follicle pool and has been shown to have limited inter- and intra-cycle variability (La Marca et al., 2007). The ultrasound assessment of AFC in the early follicular phase directly correlates with ovarian reserve (Frattarelli et al., 2000) and its decline could be a sign
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Previous losses (%)
Table II AFC reference ranges according to woman’s age as observed in 812 pregnant women with a spontaneously conceived chromosomally normal singleton fetus.
4
Grande et al.
ranges (5th, 50th and 95th percentiles).
of ovarian aging (Scheffer et al., 1999). Our study was the first to assess the AFC in a cohort of pregnant woman, since previous studies were mainly conducted in infertility patients (Chang et al., 1998; Jayaprakasan et al., 2010; Muttukrishna et al., 2005) or by histological analysis, after oophorectomy surgery and in post-portem studies (Gougeon et al., 1994; Hansen et al., 2008). Our study, one of the largest on AFC, supports a gradual AFC decline with chronological age. This finding is in agreement with the prior studies (Leidy et al., 1998; Hansen et al., 2008), although our results indicated a higher starting AFC and steeper decline. Namely, in the age range of 22 – 45 years, our series showed that the 50th centile declined from 23 to 6 follicles, while in the La Marca et al. (2011) series the observed declined was from 14 to 5 follicles, and in the Wiweko et al. (2013) series from 9 to 5 follicles. The decline for the 5th centile was from 12 to 2 follicles in our study, from 5 to 2 (La Marca et al., 2011) and from 4 to 1 follicles (Wiweko et al., 2013). However, comparisons with the current study should be made with caution as the study populations differed. Our study comprised pregnant women with spontaneous conception, while Wiweko et al. (2013) included infertile patients undergoing IVF, and La Marca et al. (2011) recruited healthy women with regular menstrual cycles attending gynaecological clinics in order to achieve a sample close to the general population. Only a small group of 65 parous women studied by Kline et al. (2004), used as control group, had a similar AFC to our pregnant population, given that the regression line decline went from 23 follicles at 22 years to 8 follicles at 42 years of age. Although the role of AFC assessment in ART is well established, scarce data are available on its association with trisomic pregnancies and its use in aneuploidy prenatal screening programs is uncertain. Our results are in
line with previous studies linking low ovarian reserve, and therefore poor oocyte quantity and quality, with trisomic offspring risk at any maternal chronological age. Thus, trisomy risk would increase with a decreasing oocyte pool size as a result of an accelerated rate of oocyte atresia (Brook et al., 1984; Kline et al., 2000). This hypothesis suggests that with a decreasing oocyte count, oocyte selection becomes impaired and the hormonal environment of the remaining oocytes may harm their integrity and quality (Warburton, 1989; Dursun et al., 2006; McTavish et al., 2007). In our study, AFC was lower than expected based on reference values in viable (trisomies 21, 18 and 13) and non-viable (other) trisomies, but not in monosomies X or triploidies, which is in accordance with the described association between distinct chromosomal anomalies and advanced maternal age. Accordingly, MoM AFC values presented a 15% reduction (0.85 MoM) in women with viable and nonviable trisomic pregnancies, while in monosomy X or triploidy normal values were observed. Subsequently, the AFC reference ranges constructed in our study allowed us to assign an ‘ovarian biological age’ to each of the pregnant women with a chromosomal abnormality. The resulting ‘ovarian biological age’ was higher than chronological age in trisomic pregnancies, as suggested by several previous studies. Firstly, women with a trisomy 21 child have been reported to present more often with a history of ovarian surgery or absence of an ovary (Freeman et al., 2000), reproducing experimental studies in mice after unilateral oophorectomy (Brook et al., 1984). Secondly, the beginning of menopause was observed to occur 1 year earlier among women with trisomic pregnancy losses (Kline et al., 2000). Thirdly, a low oocyte yield (≤4 oocytes) in women undergoing IVF was found to be associated with an increased risk of a trisomic pregnancy (Haadsma et al., 2010). Conversely, some studies have not found differences in
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Figure 1 Antral follicle count (AFC) observed in pregnant women carrying a singleton chromosomally abnormal fetus plotted against the reference
5
Antral follicle count in aneuploid pregnancies
Table III Individual maternal chronological age, AFC and AFC multiple of the median (MoM) values observed in women carrying a singleton chromosomally abnormal fetus, together with the corresponding ovarian biological age derived from the reference curves. Karyotype
Maternal age (years)
AFC (n)
AFC (MoM)
Ovarian age (years)
............................................................................................................................................................................................. Viable trisomies
Monosomies X
40
4
0.36
.45
39
8
0.66
43
47,XX+21
33
9
0.6
42
47,XX+21
34
14
0.93
36
47,XX+21
41
16
1.6
31
47,XX+21
38
19
1.46
25
47,XY+21
29
28
1.64
,16
47,XY+21
36
32
2.28
,16
47,XX+21
40
5
0.45
.45
47,XX+21
44
6
0.85
45
47,XX+21
41
9
0.9
42
47,XY+21
42
1
0.11
47,XY+21
42
9
47,XY+21
36
10
47,XX+18
44
9
1.28
42
47,XX+18
33
12
0.8
39
47,XY,+18
39
9
0.75
42
47,XY,+18
39
10
0.83
41
47,XY+13
36
12
0.85
39
Median
39
9
0.85
42 45
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Non-viable trisomies
47,XY+21 47,XX+21
.45
1
42
0.71
41
47,XY+9
39
6
0.5
47,XY+8
42
8
0.88
43
47,XY,+7
39
10
0.83
41
47,XY,+i(12)(p10)
34
7
0.46
44
47,XY+15
27
10
0.55
41
47,XY,+15
42
1
0.11
.45
47,XY,+16
34
7
0.46
44
47,XY,+16
31
21
1.31
20
47,XY+16
34
14
0.93
36
47,XY+16
33
16
1.06
31
47,XX,+16
31
18
1.12
27
47,XY+20
36
7
0.5
44
47,XX,+20
37
5
0.38
.45
47,XY,+22
33
13
0.86
38
47,XY,+22
38
20
1.53
22
47,XY+22
36
23
1.64
16
47,X,+mar,+mar
35
17
1.21
29
Median
35
10
0.86
41
45,X
30
7
0.43
44
45,X
24
15
0.78
34
45,X
40
16
1.45
31
45,X
26
18
1
45,X
37
25
1.92
,16
45,X
27
31
1.72
,16
27
mos 45,X[26]/46,XY[24]
38
8
0.61
43
Median
30
16
1.00
30.5
Continued
6
Grande et al.
Table III Continued Karyotype
Maternal age (years)
AFC (n)
AFC (MoM)
Ovarian age (years)
............................................................................................................................................................................................. Sex trisomies
47XYY
26
12
0.66
39
Triploidies
69,XXX
30
14
0.87
36
69,XXX
32
16
1.06
31
69,XXY
32
25
1.66
,16
Median
32
16
1.06
association and the potential utility of our measurements to identify mothers at higher risk of trisomic pregnancies.
Acknowledgements The authors thank Cristina Cobos, Sonia Matias and Narcis Masoller for helping in the acquisition of samples.
Authors’ roles M.G. and A.B. carried out the study design, the analysis and interpretation of data and the draft of the manuscript. A.B., V.B., J.M.J., I.S. and M.B. contributed in the acquisition of samples and revised the draft. A.B. and J.P. revised the article for important intellectual content.
Funding This study was supported by PI 11/00685. Instituto de Salud Carlos III. Fondo de Investigacio´n Sanitaria.
Conflict of interest None declared.
References Badenas C, Rodrı´guez-Revenga L, Morales C, Mediano C, Plaja A, Pe´rez-Iribarne MM, Soler A, Clusellas N, Borrell A, Sa´nchez MA´ et al. Assessment of QF-PCR as the first approach in prenatal diagnosis. J Mol Diagn 2010;12:828 – 834. Benn P, Borrell A, Cuckle H, Dugoff L, Gross S, Johnson JA, Maymon R, Odibo A, Schielen P, Spencer K et al. Prenatal Detection of Down Syndrome using Massively Parallel Sequencing (MPS): a rapid response statement from a committee on behalf of the Board of the International Society for Prenatal Diagnosis, 24 October 2011. Prenat Diagn 2012;32:1–2. Borrell A, Fortuny A, Lazaro L, Costa D, Seres A, Pappa S, Soler A. First-trimester transcervical chorionic villus sampling by biopsy forceps versus mid-trimester amniocentesis: a randomized controlled trial project. Prenat Diagn 1999;19:1138 – 1142. Borrell A, Casals E, Fortuny A, Farre MT, Gonce A, Sanchez A, Soler A, Cararach V, Vanrell JA. First-trimester screening for trisomy 21 combining biochemistry and ultrasound at individually optimal gestational ages: an interventional study. Prenat Diagn 2004;24:541 – 545. Broekmans FJ, Knauff EA, te Velde ER, Macklon NS, Fauser BC. Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metab 2007;18:58 – 65.
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AFC, inhibin B or FSH between trisomic and control pregnancies (Kline et al., 2004). The Warburton group found in a recent study differences only in FSH (Kline et al., 2011), not supporting the oocyte pool hypothesis, given that AMH was more strongly associated with chronologic age than FSH (de Vet et al., 2002). Assessing the ovarian reserve during pregnancy may have important clinical implications. Considering that AFC reflects ovarian aging, this ‘ovarian biological age’ may be a better aneuploidy risk marker than maternal age. The relationship between advanced maternal age and increased risk of fetal aneuploidy was established many decades ago (Cuckle et al., 1987). Down syndrome risk was initially estimated according exclusively to maternal age, while later this was taken as the background risk to be modified by serum maternal markers (Wald et al., 1988). Currently, cell-free fetal DNA analysis in maternal blood can also be clinically applied as a likelihood ratio to modify this background risk (Benn et al., 2012). Thus, although the use of maternal age as an exclusive criterion for increased risk of fetal aneuploidy is being abandoned worldwide, most of the Down syndrome screening models are based on refining the maternal age background risk. The strength of our study was mainly to construct the reference ranges from a large study population of 812 women, more than double the number of women compared with the previous largest series of 366 women (Wiweko et al., 2013). Furthermore, our series is the first to assess the AFC in pregnant women, demonstrating that this is feasible during the routine 11- to 13-week scan at the moment of aneuploidy risk assessment (Borrell et al., 2004). Although most of the AFC studies were focused on predicting the ovarian yield in infertile couples, our outcome measure was to determine the ‘ovarian biological age’ in women with regular cycles and spontaneous conception. We acknowledge some limitations of our study due to not performing intra- and inter-observer reliability analysis, and not using specific 3D software (SonoAVC), shown by some authors to be the best method for assessing AFC if postprocessing correction is carried out (Deb et al., 2009). Instead, we adhered to the Broekmans guidelines for 2D assessment, a well-established standardized approach to reduce AFC variability (Broekmans et al., 2010). In clinical practice, a drawback for assessing AFC is that transvaginal ultrasound is needed during the 11- to 13-week scan, when the transabdominal approach is the most commonly used. Furthermore, identifying ovaries by ultrasound during pregnancy could be challenging, particularly when the time slot assigned is limited. However, with appropriate training and motivation, sonologists can achieve this skill. In summary, current data suggest that ovarian biological age could potentially reflect a more precise background risk of fetal aneuploidy. Future studies are warranted to assess the involved complex pathophysiologic
30.5
Antral follicle count in aneuploid pregnancies
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