AMH vs. AFC as Predictive for Pregnancy Rate after ICSI View PDF

In assisted reproduction programs, a number of parameters known as ovarian reserve markers, such as serum follicle stimulating hormone (FSH) concentration, antral follicle count (AFC) and serum Anti-Müllerian Hormone (AMH) concentration, are widely used to predict ovarian responses to gonadotropin stimulation during invitro fertilization (IVF) treatment. These markers may help to decide on the initial dose and regimen of stimulation [1,2]. Among the commonly used ovarian reserve markers, AFC and AMH provide the best performance in predicting both poor and excessive ovarian response [3-5]. However, most reports consistently showed a poor predictive value of these markers on pregnancy rate in the fresh IVF cycle [4,6]. AMH, also known as Mullerian-inhibiting substance, is a dimeric glycoprotein that belongs to the transforming growth factor-beta family. It is involved in the regression of the Mullerian ducts during development of the male foetus [7]. In the adult female, AMH is exclusively produced by granulosa cells of preantral and small antral follicles, and has been shown to correlate excellently with the primordial follicle pool [8]. Hence it would serve as an ovarian reserve marker. Since AMH performs equally well, if not better, than AFC in predicting ovarian response and that it is both operator and menstrual cycle-independent, there has been a growing trend to adopt AMH assay as the first-line ovarian reserve test [2,9]. In modern-day assisted reproduction programmes, embryo cryopreservation has become an integral component. It allows the storage and subsequent usage of surplus good quality embryos in frozen-thawed embryo transfer (FET) cycles. Hence, to evaluate the outcome of an IVF cycle, it would be more logical to consider the cumulative live birth rate from the fresh and all FET cycles combined, instead of merely looking at the single fresh cycle outcome. There have been limited data on the role of these ovarian reserve markers in predicting cumulative pregnancy or live birth rates in IVF programmes. A study looked at the use of AMH on Day 6 of ovarian stimulation in predicting cumulative ongoing pregnancy outcome [10]. Yet AMH levels can be altered after commencement of ovarian stimulation [11-13].

The aim of this work is to compare the predictive value of Anti-Müllerian Hormone (AMH) and antral follicle count on fertilization rate (FR), implantation rate, blastocyst development, embryo quality, chemical pregnancy, clinical pregnancy and ongoing pregnancy after ICSI.

This quasi-experimental study was conducted in the Department of Obstetrics and Gynecology, El-Minia Infertlity center Faculty of Medicine, El-Minia University and two private centers during the period from June 2016 to June 2018 after being approved by the department ethical Committee. The study population included 56 subjects aged between 25 and 42 years, enrolled for intra cytoplasmic sperm injection (ICSI) program.

The inclusion criteria were:

• First stimulation cycle for intracytoplasmic sperm injection (ICSI).
• A long protocol for the use of a Gonadotrophin releasing hormone agonist (GnRHa).
• The presence of both ovaries.
• Regular menstrual cycles (cycle length of 25 to 35 days duration).
• No evidence of endocrine disorders (normal TSH, prolactin, testosterone, and androstanidione).
• A BMI ranging from 18 to 25 Kg/m2
• Not on hormone therapy for previous 3 months.
• No history of ovarian surgery.

Serological markers for hepatitis B, C, and HIV were negative Antral follicle count and hormonal assay Baseline hormone profiles including serum levels of FSH, LH, and AMH were determined on day 3 of the previous cycle. The antral follicle count measurements were performed by the same sonologist on days 3-5 of the menstrual cycle. Antral follicles within the bilateral ovaries between 2-6 mm in diameter were recorded. An 8 MHZ transvaginal probe was utilized for all examinations. Serum FSH and LH were measured using a specific immumetric assay kit (Immulite; Diagnostic products corporation, Los Angeles CA, USA). The minimal detection limits for FSH was 0.1 IU/L, and the intra- and inter-assay coefficients of variation for the FSH assay were 6.6% and 7.8%, respectively. The detection limit and intra- and inter-assay coefficients of variation for the LH assay were 0.1 IU/L and 6.5/7.1%, respectively. Measurement of serum AMH levels was performed using AMH/MIS Elisa kit (Diagnostic systems Lab, Webster Texas, USA). The Minimal detection limit and intra- and inter-assay coefficients of variation for the AMH assay were 0.017 ng/ml and 5% and 8%, respectively.

Ovarian Stimulation Protocol

All patients were treated with long protocol for ovarian stimulation. In the long protocol, pituitary down regulation was achieved by administering luprolide acetate (Lupride 4 Sun pharma) 0.5 mg subcutaneously daily from day 21 of the previous menstrual cycle. Ovarian stimulation was effected with exogenous gonadotrophin in the form of recombinant follitrophin β (racy-FSH puregon, Organon, Germany) or menotrophin (LG IVF.M, LG Life sciences, Korea) from day 2 of the menstrual cycle. The starting dose of gonadotrophin for subjects under 35 years age was 150 IU/day and for subjects over 35 years age was between 225 and 300 IU/day. After 7 days of stimulation, follicular growth was assessed by transvaginal ultrasound with 8 MHZ probe. The dose of recombinant FSH was adjusted according to the ovarian response. Ovulation was induced with 10,000 IU of hCG (Koragon Ferring pharma, India) when at least 3 follicles attained the size of more than 17 mm in diameter. Transvaginal oocyte retrieval was performed under ultrasound guidance 36 hours after hCG administration, and the number of retrieved oocytes was recorded. ICSI was performed on the retrieved oocytes. An oocyte count less than 4 and absence of follicular growth with controlled ovarian hyper stimulation were considered as poor ovarian response. An oocyte count of 4 or more was considered as good ovarian response.

The primary outcome measures of the study were:

• Number of oocytes retrieved and the ovarian response,
• To compare AMH and AFC as predictors of pregnancy rate.

Statistical analysis was performed using SPSS software trail version 20.0. Fisher's exact test was used to examine the difference in categorical variables. Values are presented as mean ± SD. Students ‘t’ test was performed to compare different groups. Pearson correlation coefficients were calculated to explore the relationship between the measured parameters. Multivariate logistical regression analysis was used to test the association between poor response (oocyte count < 4) and normal response with measured parameters. For all statistical analysis, P < 0.05 was considered significant.

The mean age (years) of the subjects studied was 34.61 ± 3.62. The mean AMH levels were 1.79 ± 1.72 ng, and mean AFC was 8.57 ± 5.16. The mean FSH levels were 5.60 ± 3.64. AMH and AFC levels were higher in normal responders. There were statistically significant positive correlations between the number of retrieved oocytes and AMH (r = 0.543, P < 0.01) followed by AFC (r = 0458, P < 0.01). Statistically significant but inverse correlations between the number of oocytes and age (r = -0314, P < 0.01) were observed. No correlation was identified between the number of retrieved oocytes and FSH (r = -0.179, P > 0.05). Patients were divided into 2 groups depending on the number of oocytes retrieved as poor (<4 oocytes) and normal (more than 4 oocytes) responders. There were 33 normal responders and 23 poor responders. Statistically significant differences were observed in mean AMH levels between normal responders 2.19 ± 1.74 ng/ml and poor responders 1.22 ± 1.55 ng/ml (P = 0.036). Similarly, the mean AFC was 10.42 ± 5.56 in normal responders and 5.91 ± 4.31 in poor responders (P = 0.002). The mean no of oocytes retrieved in normal responders was 12.27 ± 6.06 and 2.22 ± 1.24 in poor responders (P = 0.001). When multiple regression analysis was used for prediction of ovarian response, AMH levels and AFC were found to be independent predictors of ovarian response β-coefficient [± SE] for AMH was 1.618 ± 0.602, P = 0.010 and for AFC, it was 0.528 ± 0.175, (P = 0.004).

In this study, the role of AMH and AFC were evaluated and compared for predicting pregnancy rate to controlled ovarian stimulation with gonadotropin. Out of the 56 subjects recruited for the study, 33 subjects had normal ovarian response, 22 had poor ovarian response, and 1 subject had empty follicular syndrome. The mean AMH levels and AFC were significantly high in normal responders compared to poor responders. Similar observations were made in other studies [2,7]. Statistically significant positive correlations were found for AMH and AFC with regard to ovarian response (P = 0.01). Similarly, significant inverse correlations were found with respect to age (P = 0.01). This age-related decline in ovarian reserve was also observed in other published studies [4,14]. Statistically significant correlations were not found with respect to other studied parameters [14] like FSH and estradiol (P = >0.05). These observations are in accordance with the results of other studies conducted recently [3-5,15]. In two recent studies, AMH levels were correlated with ovarian response, oocyte quality, and cycle cancellation [3,15]. In the first study, the mean AMH levels were 1.26 ng/ml, and in the second study, the mean AMH levels were 1.66 ng/ml. In the present study, the mean AMH levels were 1.22 ng/ml and 2.19 ng/ml in poor and normal responders, respectively. This study shows that fertility is preserved even at lower AMH levels in Indians compared to their western counterparts. Various other studies also correlated AMH levels and AFC with oocyte count and quality [2,9,16-18]. When multiple regression analysis was used to compare the measured parameters, both AMH and AFC were found to be independent predictors of ovarian response. The present study indicates that AFC was a better predictor of ovarian response compared to AMH (P = for AFC and 0.01 for AMH). This is an important observation as AMH is an expensive test available at few places, whereas AFC counts are measured routinely by infertility consultants. This study demonstrates that AFC can be used as a surrogate for expensive AMH estimation, which can be used for females above 35 years age where it is said to be more reliable for predicting ovarian reserve and pregnancy outcome [19-23]. Recent studies have revealed AMH and AFC as predictors of primordial follicle pool and excessive response to controlled ovarian hyperstimulation [5,24]. Currently, most IVF clinicians determine starting doses of gonadotropin in the first cycle of IVF, depending principally on age and basal FSH levels [17]. Our study indicates that AMH and AFC are also good predictors of ovarian response to controlled ovarian stimulation. The conventional ultrasound assessment for the determination of ovarian reserve is operator-dependent as it involves subjective interpretation of the images. This can be eliminated by automated evaluation of the antral follicle size and number and thereby improve the outcome. At the present time, one of the principal impediments to the more widespread use of AMH to predict ART outcome is lack of standardization of AMH assay results. Even though AMH and AFC are good predictors of ovarian reserve, they should not be used for excluding subjects from ART programs. They are useful for counselling the infertile couples about realistic outcome of the procedure. Creating realistic expectation is very important in maintaining patient confidence and satisfaction. These tests may also help the clinicians to make adjustments in the starting dose of gonadotropin, thereby preventing excessive stimulation and ovarian hyper stimulation syndrome.

This study reveal that both AMH and AFC are good predictors of pregnancy rate to controlled ovarian hyper stimulation.

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