Scholarly article on topic 'Follicular fluid antimullerian hormone (AMH) does not predict IVF outcomes in polycystic ovary syndrome patients'

Follicular fluid antimullerian hormone (AMH) does not predict IVF outcomes in polycystic ovary syndrome patients Academic research paper on "Basic medicine"

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{"Follicular fluid" / "Antimullerian hormone" / AMH / IVF / PCOS}

Abstract of research paper on Basic medicine, author of scientific article — Bushra Abu-Fakher, Faizeh Al-Quobaili, Marwan Alhalabi

Abstract Introduction AMH, is a member of the transforming growth factor (TGF-β) produced by granulosa cells with the highest expression being in small antral follicles. Our objective is to evaluate the predictive value of follicular AMH levels as an indicator of IVF outcomes in PCOS patients. Material and methods A total of 63 patients undergoing the IVF trail were recruited for this prospective case-control study. The patients were classified into three groups: Group I: 43 patients with Polycystic ovary syndrome (PCOS). Group II: 20 normo-ovulatory patients were recruited for the IVF trail because of the male infertility factor (control group). Group III: consists of 33 patients from group I recruited to determine the effect of controlled ovarian hyperstimulation (COH) on serum AMH levels. Serum AMH levels were performed on day 3 of the menstrual cycle whereas the follicular fluid AMH level has been evaluated at the time of oocyte retrieval by enzyme linked immune-sorbent assay (AMH Gen II ELISA kit, Beckman Coulter, Inc. USA). AMH concentrations were adjusted to its protein content, to avoid possible bias due to FF volume variability. The relationship between follicular fluid AMH (FF AMH) levels and the number of oocytes retrieved, number of mature oocytes, number of embryos, fertilization and clinical pregnancy rate were assessed. The demographic data were similar between two groups as regards age and BMI. Results Both serum AMH levels and FF AMH levels were significantly higher in PCOS patients than in the controls, the values for serum levels were (5.07±3.39ng/ml, 3.07±2.09ng/ml) respectively and for follicular fluid levels were (67±50.7ng/g∗, 38.5±44.4ng/g) respectively. Serum AMH levels showed a positive correlation with oocytes retrieved and mature oocyte number, but there was no significant correlation with good quality embryo number, fertilization rate or clinical pregnancy rate in PCOS patients. On the other hand FF AMH levels do not show correlation with any of the IVF outcomes mentioned above in PCOS patients. Interestingly, the interval change in serum AMH levels between baseline AMH levels and levels after COH had a significant predictive value of pregnancy. Conclusions Serum AMH levels can predict the number of the retrieved oocytes and mature oocytes, whereas FF AMH may not be a valuable predictor for IVF outcomes in PCOS patients.

Academic research paper on topic "Follicular fluid antimullerian hormone (AMH) does not predict IVF outcomes in polycystic ovary syndrome patients"

Middle East Fertility Society Journal (2013) 18, 110-114

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

ORIGINAL ARTICLE

Follicular fluid antimullerian hormone (AMH) does not predict IVF outcomes in polycystic ovary syndrome patients

Bushra Abu-Fakher a *, Faizeh Al-Quobaili b, Marwan Alhalabi c,d

a Faculty of Pharmacy, Damascus University, Damascus, Syria

b Department of Clinical Biochemistry and Microbiology, Faculty of Pharmacy, Damascus University, Syria c Department of Embryology and Reproductive Medicine, Faculty of Medicine, Damascus University, Syria d Assisted Reproduction Unit, Orient Hospital, Damascus, Syria

Received 28 October 2012; accepted 9 December 2012 Available online 4 February 2013

Abstract Introduction: AMH, is a member of the transforming growth factor (TGF-b) produced by granulosa cells with the highest expression being in small antral follicles. Our objective is to evaluate the predictive value of follicular AMH levels as an indicator of IVF outcomes in PCOS patients.

Material and methods: A total of 63 patients undergoing the IVF trail were recruited for this prospective case-control study. The patients were classified into three groups: Group I: 43 patients with Polycystic ovary syndrome (PCOS). Group II: 20 normo-ovulatory patients were recruited for the IVF trail because of the male infertility factor (control group). Group III: consists of 33 patients from group I recruited to determine the effect of controlled ovarian hyperstimulation (COH) on serum AMH levels. Serum AMH levels were performed on day 3 of the menstrual cycle whereas the follicular fluid AMH level has been evaluated at the time of oocyte retrieval by enzyme linked immune-sorbent assay (AMH Gen II ELISA kit, Beckman Coulter, Inc. USA). AMH concentrations were adjusted to its protein content, to avoid possible bias due to FF volume variability. The relationship between follicular fluid AMH (FF AMH) levels and the number of oocytes retrieved, number of mature oocytes, number of embryos, fertilization and clinical pregnancy rate were assessed. The demographic data were similar between two groups as regards age and BMI.

Results: Both serum AMH levels and FF AMH levels were significantly higher in PCOS patients than in the controls, the values for serum levels were (5.07 ± 3.39 ng/ml, 3.07 ± 2.09 ng/ml) respectively and for follicular fluid levels were (67 ± 50.7 ng/g , 38.5 ± 44.4 ng/g) respectively. Serum

* Corresponding author. Tel.: +963 932749900.

E-mail address: ph.bushra@outlook.com (B. Abu-Fakher).

Peer review under responsibility of Middle East Fertility Society.

KEYWORDS

Follicular fluid;

Antimullerian hormone;

1110-5690 © 2013 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mefs.2012.12.005

AMH levels showed a positive correlation with oocytes retrieved and mature oocyte number, but there was no significant correlation with good quality embryo number, fertilization rate or clinical pregnancy rate in PCOS patients. On the other hand FF AMH levels do not show correlation with any of the IVF outcomes mentioned above in PCOS patients. Interestingly, the interval change in serum AMH levels between baseline AMH levels and levels after COH had a significant predictive value of pregnancy.

Conclusions: Serum AMH levels can predict the number of the retrieved oocytes and mature oocytes, whereas FF AMH may not be a valuable predictor for IVF outcomes in PCOS patients.

© 2013 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Polycystic ovary syndrome is the most frequent cause of anovulatory infertility and hyperandrogenism in young women (1). It affects upto 10% of reproductive age women (2). PCOS is characterized by a clustering of hyperandrogenism, hyperin-sulinemia, hypersecretion of LH, menstrual dysfunction, hir-sutism, infertility and pregnancy and neonatal complications (3). Although PCOS patients are typically characterized by producing an increased number of oocytes, they are often of poor quality, leading to lower fertilization, cleavage and implantation rates, and a higher miscarriage rate in PCOS patients undergoing IVF treatment (4).

AMH is a glycoprotein of the transforming growth factor-beta (TGF-b) super family. AMH is produced by granulosa cells from 36 weeks of gestation until menopause, with the highest expression being in small antral follicles. AMH production gradually declines as follicles grow, once follicles reach a size at which they are dominant it has largely disappeared. Its removal from these larger follicles appears to be an important requirement for dominant follicle selection and progression to ovulation as AMH has an inhibitory role in the ovary, reducing both primordial follicle initiation and follicle sensitivity to FSH by the inhibition of aromatase. It is for this reason that AMH is a focus of interest in polycystic ovary syndrome (5).

Women with PCOS have elevated serum and FF AMH levels versus those of normal controls (6). Recent study suggests that increased FF AMH in women with PCOS may have harmful consequences on oocyte quality and maturation, via an unclear molecular mechanism, but does not have an effect on pregnancy rates (7), whereas other investigations reveal that women with PCOS who have lower FF AMH levels have similar rates of oocyte maturation, fertilization and embryonic development compared with normo ovulatory women (8).

To determine whether AMH could play a role in the success rate of IVF- embryo transfer, this study aims to confirm the relationship between serum and intrafollicular AMH levels, and to determine the relationship of intrafollicular AMH to IVF outcomes in PCOS patients and normal ovulatory women.

2. Materials and methods

A prospective case - control study was performed in a total of 43 infertile PCOS patients and 20 normo-ovulatory patients. The diagnosis of PCOS was based on Rotterdam criteria 2003, the association of at least two of the three following criteria:

1) Ovulatory disturbance, mainly oligomenorrhea or amenorrhea.

2) Hyperandrogenism as defined either clinically by hirsut-ism (modified Ferriman and Gallwey score > 6), or severe acne/seborrhea, and/or biologically by a testosterone serum level greater than 0.7 ng/ml and/or D4-andro-stenedione greater than 2.2 ng/ml.

3) More than 12 follicles in the 2-9 mm range in each ovary with peripheral distribution at ultrasound and/or an ovarian volume higher than 10 ml were found. Ultra sound examination was performed with a 7.5-MHz transvaginal transducer.

All the control population underwent the IVF trail because of the male factor. Exclusion criteria for the controls were a history of menstrual disturbances, absence of one of the ovaries, abnormal serum levels of prolactin or androgens (i.e. serum testosterone above 0.7 ng/ml and/or D4-androstenedione above 2.2 ng/ml) and any hormonal treatment during the 3 months before the study.

The patients followed a long protocol for controlled ovarian hyperstimulation which was initiated with the oral contraceptive pill (OCP) on day three or five of the cycles and continued with pituitary downregulation by a gonadotropin-releasing hormone (GnRH) agonist consisting of 0.05 mg of buserelin in the mid-luteal phase (day 21) of the cycle, given daily until the day of human chorionic gonadotropin (hCG) administration.

On day 2-3 of the new cycle, ovarian stimulation was started with an injection of recombinant FSH or human menopausal gonadotropin (hMG). The gonadotropin dosage was adjusted according to the follicular growth, which was monitored by ultrasound. The patients were given 10 000 IU of hCG when at least three follicles became more than 1718 mm. The oocytes were retrieved 35 h after hCG administration and IVF/ICSI followed by embryo transfer was performed 3 days after oocyte retrieval.

Blood samples were collected on day three of the menstrual cycle. On the day of oocyte retrieval, the follicular fluid was aspirated from all follicles (14-20) mm, and then it was centri-fuged at 4000 rpm for 10 min and stored at -80°C. All follic-ular fluid samples were examined on the same day to measure AMH levels using enzyme linked immunosorbent assay (Beck-man Coulter, Inc. USA). AMH concentrations were adjusted to its protein content, to avoid possible bias due to FF volume variability, by the Bradford assay. The assay was performed in a 96 well plate using Bradford's reagent. Suitable dilutions of bovine serum albumin (BSA) 1 ig/ml were used as standard solutions. All FF samples were diluted 250 times, the absor-bance of standard and unknown solutions were determined using a spectrophotometer. The concentrations of AMH in fol-

licular fluid were presented as nanogram per gram (ng/g) of protein.

3. Statistical analysis

The data were analyzed with MedCalc® v12.2.1. Data were expressed as mean ± standard deviation. Comparison of the means was performed by the Mann-Whitney U test while the degree of association between continuous variables was calculated by the Spearman's correlation coefficient.

A p value <0.05 was considered statistically significant.

4. Results

Demographic characteristics of each group (age, BMI, serum AMH, FF AMH, retrieved oocyte number, mature oocyte number, embryos number and fertilization rate are shown in Table 1.

As expected PCOS patients had significantly higher serum AMH levels than controls (5.07 ± 3.9 ng/ml, 3.07 ± 2.09 ng/ ml) respectively (p < 0.01). On the other hand FF AMH levels were significantly higher in PCOS patients compared to the controls (67 ± 50.7 ng/g, 38.5 ± 44.4 ng/g) respectively (p < 0.05). Serum AMH levels were negatively correlated to age in the control group (r = —0.45, p = 0.04) but not in PCOS patients. (r is Spearman correlation coefficient).

In PCOS patients, serum AMH levels were positively correlated to the number of oocytes retrieved (r = 0.39, p = 0.01) and the number of mature oocytes (r = 0.334, p = 0.03) (Figure 1) but not to the number of total embryos or the fertilization rate, whereas follicular fluid AMH levels were not significantly correlated to any of the IVF outcomes mentioned above.

Similar results were found in normo - ovulatory women; serum AMH levels were positively correlated to oocytes retrieved (r = 0.513, p = 0.02) mature oocytes (r = 0.615, p = 0.002) (Figure 1) and embryo number (r = 0.625, p = 0.002).

FF AMH levels were not significantly correlated to any of the IVF outcomes as well. Both serum and FF AMH levels had not a significant predictive value of pregnancy. But interestingly the interval change between baseline serum AMH levels and levels after controlled ovarian hyperstimulation (values are shown in Table 2) had a significant predictive value of pregnancy.

The predictive potency of the AMH interval change was tested by the Receiver operating characteristic (ROC) procedure. The area under the curve was 0.697. Several cutoff values

ft PCOS O Control ^-PCOS '"--Control

12.50-

E 10.00-

o.oo- o

—I-1-1-1-1-1—

0 10 20 30 40 50

Mil oocyte number

Figure 1 Relationship between serum AMH on day three and the number of mature oocytes in both PCOS and control groups.

of AMH interval change were analyzed in terms of specificity and sensitivity from the ROC curve data (Figure 2).

Table 3. Showed that the best adjustment between specificity (92%) and sensitivity (54%) was obtained with a cutoff value of 54.7%.

5. Discussion

The finding of increased AMH levels in both follicular fluid and the serum of PCOS patients compared with controls can be explained with the fact that PCOS patients have increasing number of small antral follicles which are the major sites of producing AMH and further more by the finding of Pellat et al. who demonstrated that AMH production was on average 75 times higher per granulosa cell from anovulatory polycystic ovaries than from normal cells (9). Our finding is in agreement with the study of Fallat et al. who reported that the level of AMH in the follicular fluid and serum of patients with PCOS undergoing IVF was significantly higher than that in patients with endometriosis or pelvic adhesions (10). We found a negative correlation between age and serum AMH only in the control group whereas FF AMH levels remain unaffected by age, this finding is in agreement with both Das M et al. (11) and Pigny et al. (6).

We found that serum AMH levels in PCOS patients can predict oocyte quantity and quality (positive correlation with mature oocytes) and this finding is in accordance with both Arabzadeh et al. (12) and Aleyasin et al. (13).

No correlation between FF AMH and any of IVF outcomes mentioned before were found in the PCOS group, this is partially in agreement with Desforges who suggests that increased FF AMH in women with PCOS may have harmful consequences on oocyte quality but does not have an effect

Table 1 Clinical and biological features of patients.

Controls PCOS patients p-value

(n = 20) (n = 43)

Age (year) 29.2 ± 6.2 29.2 ± 5.2 NS

BMI (kg/m2) 25.5 ± 3.9 26.3 ± 6.2 NS

sAMH (ng/ml) 3.07 ± 2.09 5.07 ± 3.39 0.004

FF AMH (ng/g) 38.5 ± 44.4 67 ± 50.7 0.02

Retrieved oocyte no. 14.4 ± 8.2 20.1 ± 11.2 0.05

Mature oocyte no. 10.1 ± 5.3 13.3 ± 8.6 0.04

Total embryos no. 8 ± 4.3 13.2 ± 9.1 0.04

Fertilization rate 84.2 ± 19.4 83.8 ± 21.7 NS

*NS = non-significant; Values are expressed as the mean ± SD.

Table 2 Serum AMH interval change.

Baseline serum AMH levels Serum AMH after COH Interval change

(ng/ml) (ng/ml) (%)

PCOS patientsa (n = 33) 5.4 ± 3.4 1.9 ± 1.3 60.8 ± 20.9

Controlsa (n = 20) 3.07 ± 2.09 1.04 ± 1.06 68.7 ± 16.6

a mean ± SD.

Figure 2 ROC curve for AMH interval change.

Table 3 ROC curve data.

AMH interval change (%) Specificity (%) Sensitivity (%)

36.8 20.8 100

47.1 37.5 96

54.7 54.1 92

68.4 58.3 48

80.4 91.6 28

on pregnancy rates (7). Mashiach found that women with PCOS who have lower FFAMH levels have similar rates of oo-cyte maturation, fertilization, and embryonic development compared with ovulating women (14). On the other hand, conflicting results were obtained from Pabuccu 2009 who demonstrated better fertilization, implantation and clinical pregnancy rates in the PCOS group with highest FFAMH concentration compared with any other group with a lower concentration (8).

In normo-ovulatory women, we found that serum AMH levels could be a good predictor for oocyte quantity and quality (positive correlation with maturation rate) and furthermore for embryo number, and this is in agreement with Vanrooij et al. 2002 who demonstrated that serum AMH levels are a good predictor of retrieved oocytes (15) and with Arabzadah 2010 who found that serum AMH levels can predict both the quantity and quality of oocytes (12). FF AMH levels do not relate to any of the IVF outcomes in PCOS patients and this is in agreement with Yilmaz 2012 who showed that there is no significant correlation between FF AMH and IVF out-

comes in patients underwent IVF for male infertility or unexplained infertility(16). Fanchin et al. suggest that FF AMH concentrations are only strongly and positively associated with oocyte quality and implantation rates, but not with fertilization rate, and embryo morphology in normal ovulation (17) and this conflict can be explained by the differences in the patients groups since Fanchin et al. studied non PCOS infertile patients who underwent the IVF trial.

There are conflicting results about the relation between AMH levels and pregnancy, the most recent studies demonstrate that Clinical pregnancy rates were lower in the high AMH group than that of the low and average groups but this difference was only close to statistical significance Xi et al. 2012 (18), whereas Yilmaz 2012 found that FF AMH levels can predict the recovery of oocytes but not oocyte quality, embryo quality or pregnancy in non-obese non-hyperandrogene-mic PCOS patients (16). Desforges 2009 demonstrated that the AMH levels were significantly lower in patients who began a pregnancy (7).

So, we concluded that AMH may not be a valuable predictor for outcomes' success in PCOS patients and normo-ovula-tory women undergoing IVF. Other randomized control studies are needed.

6. Conflict of interest

None. References

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