International Journal of

 Introduction 
   Recognition and selection of the best oocyte and embryo can improve IVF outcome. Follicular fluid is composed of substances which are secreted by granulosa cells, theca cells and blood flow. It is a suitable environment for oocyte growth and development. Biochemical characteristics of the follicular fluid play an important role in the prediction   of   oocyte   quality,   fertilization   and ultimately   the   embryo   quality   in   noninvasive methods (1, 2). Anti-mullerian hormone (AMH) is one member of transforming growth factor-β (TGF-β) (2-5). AMH plays a fundamental role in gonadal differentiation during fetal period and inhibits the formation of mullerian ducts in male fetus (2, 6-8). AMH is secreted by the ovarian granulosa cells into blood flow and follicular fluid in adult female, although its concentration is much higher in the follicular fluid (2,4,6). AMH production is independent of FSH and inhibits FSH-induced follicular growth (9-11). It also has a direct autocrine-paracrine effect on the granulosa cells, oocyte function and embryo quality (5, 12-14). Determination of oocyte and embryo quality are one of the most important goals of embryologists in human IVF. Several methods are employed for determining oocyte and embryo quality(1). Follicular fluid anti-mulerian hormone (FF AMH) level is probably a marker of the qualitative and quantitative activity of granulose cells (13). Although some studies have showed the relationship between the level of serum AMH , and quality of oocyte and embryo (9, 11, 12), there are few studies about the relationship existing between these factors and FF AMH level (13). Our study was designed to investigate the association between FF AMH level and successful fertilization and embryo quality in IVF cycles. Based on FF AMH level, we can choose one or two embryos of high viability and thus improve IVF outcome.
 
Materials and methods
 
     The study was conducted at a reproduction center affiliated to a medical university between February and January 2009. A total of 62 infertile patients, who underwent IVF, participated in this prospective study. The inclusion criteria were: female age <35 years old, presence of both ovaries in ultrasonography, BMI <25 kg/m², day 3 FSH<10 IU/ml, and the duration of menstrual cycle between 25-35 days.  Patients with PCOD, history of pelvic surgery, endometriosis, endocrine disorders and couples with male factor who were candidate for ICSI (patients with immotile sperm, azospermia, normal morphology of sperm<4% and frozen-thawed sperms) (15) were not enrolled in this study. Also, patients whose follicular fluid was bloody during the oocyte retrieval were excluded from the study. Before initiating the treatment, venipuncture for assay of FSH, estradiol (E2), and transvaginal ultrasound scan were also performed on the third day of the menstrual cycle.
     This study was approved by the ethics committee of Research and Clinical Center for Infertility affiliated to Shahid Sadoughi University of Medical Sciences. Furthermore, all patients were required to sign a written consent before the initiation of the treatment cycles.
 
Assay
     All transvaginal ultrasonographic evaluations were performed by a single investigator, using a conventional two-dimensional ultrasound (HS-400, Honda, Japan) equipped with a 7.5 MHz vaginal transducer. FSH concentrations were measured by competitive immunoassay (IDCS, Korbach, Germany), intra-assay and inter-assay coefficients of variation were 6% and 6.8% respectively. E2 concentrations were measured using an enzyme-immunoassay kit (DRG, Marburg, Germany), intra-assay and inter-assay coefficients of variation proved to be 6.3% and 6.4% respectively. Measurement of FF AMH levels was performed using AMH/MIS enzyme-linked immunosorbent assay kit (Beckman Coulter Immunotech Com., Fullerton, CA).
 
Treatment protocol
     All of the patients were treated with a long protocol for ovarian stimulation. For pituitary suppression, the patients were treated with daily administration of 0.5 mg/day buserelin SC (Superfact, Aventis, Frankfurt, Germany) which started in the luteal phase of menstrual cycle. When desensitization was occurred, as evidenced by plasma E₂ levels of ≤50 pg/ml and the absence of ovarian cyst on transvaginal ultrasound examination, buserelin was reduced to 0.25 mg/day and continued until the day of hCG administration. The COH (controlled ovarian hyperstimulation) was initiated with recombinant FSH (Gonal F, Serono, Aubnne, Switzerland) or HMG (Menogon, Ferring, Pharmaceuticals, Germany) 150 IU/day on the day 2 of menstrual cycle. Ovarian response was monitored by serial ultrasound examinations and the evaluation of serum E2 levels, then gonadotropin doses adjustment was done as required. Urinary human chorionic gonadotropin (Pregnyl, Organon, Oss, the Netherlands) 10000 IU was administered when at least three follicles reached a mean diameter of 18mm.
     Oocyte retrieval was performed by the transvaginal ultrasound guided approach, 34-36 hours after the hCG injection. Each patient’s follicular fluid sample was collected from one follicle with a diameter greater than 17 mm. Only the first follicle of ovary was selected and the needle was washed before the puncture of the remaining follicles. Follicular fluid of the first selected follicle was separated from the cumulus-oocyte complex and then was centrifuged.
     Each of the centrifuged follicular fluid was freezed at -80°c until the samples were all completed. Then, the AMH level of every respective follicular fluid was measured and conventional IVF was performed specifically on the oocyte obtained from the follicular fluid in a separate culture dish. The fertilization of each oocyte was assessed using a microscope 18-20 hours following IVF and the fertilization was confirmed through pronuclei detection. Also the quality of embryo was determined in fertilized oocyte 48 hours after IVF and was scored based on the shape, number and fragmentation of blastomers (16). Score≥18 was decided to indicate good quality, 15-17 fair quality and <15 poor quality in our center. Based upon fertilization of oocytes, the patients were divided into two groups. Patients with fertilized oocytes were defined as group I and non-fertilized oocytes as group II. FF AMH level and quality of embryos were compared in both groups.
 
Table I. Comparison of the patient’s characteristics in fertilized and non-fertilized groups.


Table  II. Etiology of infertility in fertilized and non-fertilized groups.


Discussion
 
     In the present study, the correlation between FF AMH level and fertilization, and embryo quality was investigated. According to the findings of this study, FF AMH level in fertilized group was higher than that in non-fertilized group and there was a significant difference between the two groups in this regard. Also, the FF AMH level was significantly higher in good quality embryos.
     Similarly, Takahashi et al investigated FF AMH level in patients who had undergone IVF. Their study indicated that the concentration of FF AMH in fertilized group was higher than that in non-fertilized group. They concluded that FF AMH level proves to be an important indicator of fertilization   (17).   Mashiach   et   al   reported   a positive relationship between FF AMH level and
 
embryo quality in women who had undergone IVF. Mashiach’s study was performed on PCOD women while our study was done on women with normal
menstrual cycles (18). Jancar et al also showed that FF AMH level in modified natural cycles were higher than those in COH cycles. In contrast with our results, they found no significant difference between AMH level and embryo quality in modified natural cycles (19). One reason for the differences in correlation between FF AMH level and embryo quality in different studies is probably the variety of embryo scorings in IVF centers. In two studies, FF AMH level was significantly higher in women who were conceived with IVF (20, 21). Fanchin’s investigation failed to find any significant difference between FF AMH level and embryo scores. However, they showed that higher FF AMH level was associated with higher implantation rate (20). Cupisti and Lee’s studies found no correlation between the number of follicles and retrieved oocytes with FF AMH level (22, 23). We were not able to prove the existence of any such relationship because we had selected only one follicle in each patient.

Conclusion

Our results indicate that there exists a positive correlation between FF AMH level and fertilization, and embryo quality. Therefore, we can make use of FF AMH level as an indicator in IVF outcome.

 

Acknowledgement

     The authors are grateful to Farimah Shams for her assistance in statistical analysis and the laboratory staff of the Research and Clinical Center for Infertility for their assistance.