Anti-mullerian Hormone and Vitamin D as a predictor of Ovarian reserve and Ovarian response in Infertile women undergoing IVF

 

Hiba H. Kadhim, Salman A. Ahmed

Department of Chemistry, College of Science, Al-Nahrain University, Baghdad-Iraq.

*Corresponding Author E-mail: hiba.hyder26@yahoo.com

 

ABSTRACT:

Aim: This study was aimed to measure AMH and 25(OH)D as a predictor of ovarian reserve among infertile females undergoing IVF in Iraq. Objective: To estimate anti-mullerian hormone as a predictor of fertility potential in terms of ovarian reserve and ovarian response reflected by antral follicles and mature oocyte counts in response to stimulation in in vitro fertilization (IVF) females. Materials and Methods: This prospective cohort study consisted of 60 women (mean age 26.67 years) undergoing IVF at Al-Amal Fertility Center in Baghdad. Blood withdrawal for Anti-mullerian hormone, vitamin D, FSH and E2 measurement was performed in all the patients and the number of oocytes were recorded. Results: Results showed that the ovarian response was better with younger age (<4, 4-8, 9-16 and >16 There was a significant positive association between ovarian response in terms of total number of oocytes and AMH levels (<4, 4-8, 9-16 and >16 oocytes there was no association between ovarian response in terms of total number of oocytes and vitamin D, FSH and E2 Moreover, Correlation coefficient revealed that the number of mature oocytes showed strong positive correlation with the AMH levels (r=5.27, p=0.001). The total number of oocytes was inversely associated with age There was a significant positive association between ovarian response in terms of the total number of oocytes and AMH levels there was no association between ovarian response in terms of the total number of oocytes with 25(OH)D FSH and E2 implying that AMH can be used as a good predictor of ovarian reserve and ovarian response. Conclusion: Anti-mullerian hormone can be used in IVF programs as a good predictor of ovarian reserve and ovarian response.

 

KEYWORDS: Anti-mullerian hormone, Ovarian reserve, IVF, vitamin D, Infertility.

 

 


INTRODUCTION:

Anti-mullerian hormone (AMH) also known as mullerian-inhibiting substance or mullerian inhibiting factor is a glycoprotein dimer composed of two 72 KDa monomers linked by disulfide bridges (1) AMH is an essential marker of ovarian reserve and ovarian ageing (2), its clinical applications include predicting the response to ovarian stimulation during in vitro fertilization (IVF) (3).

 

 

 

The hormone acts through AMHR-II, a specific type II receptor, and contributes to maintaining the oocyte pool by inhibiting recruitment and subsequent maturation of primordial follicles (4). AMH belongs to the transforming growth factor-β super family and binds to AMH receptor 2. (5). AMH is produced by ovarian granulosa cells in female. After puberty, when menstrual cycling begins, circulating AMH slowly decreases throughout life and becomes undetectable at menopauses (6). AMH continues to be expressed in the growing follicles in the ovary until they have reached the size and differentiation state at which they are to be selected for dominance. In humans, this occurs at the antral stage when the follicle size is 4-6 mm. (7). AMH seems to act only in the reproductive organs (8). Vitamin D is a lipid-soluble vitamin synthesized primarily by the skin on exposure to ultraviolet light. Few foods contain vitamin D, consequently, dietary vitamin D contributes only 10%–20% to overall body stores. Vitamin D levels exhibit seasonal fluctuations due to changes in sunlight exposure throughout the year (9). Dietary and dermally produced vitamin D are biologically inactive and require enzymatic conversion to active metabolites (10). In the past, clinical interest in vitamin D has mostly been centered on its critical role in calcium homeostasis and bone metabolism. More recently, discoveries of the vitamin D receptor (VDR) in a variety of other organ tissues suggest a much wider role for vitamin D. It is converted enzymatically in the liver to 25 hydroxyvitamin D (25OH-D), the major circulating form, and then in the kidney to 1, 25-dihydroxyvitamin D (1, 25OH-D), the active form of vitamin D (10). It has been implicated in obesity, immune function, metabolic syndrome, and cardiovascular disease (10), as well ovarian function and pregnancy outcomes (11). Hypovitaminosis D has also been associated with primary ovarian insufficiency in humans (12). Rodent models have demonstrated that vitamin D deficiency can be a cause of infertility (13). A recent metaanalysis found that women with adequate vitamin D levels have increased chances of achieving pregnancy after assisted-reproductive technology (ART) and a higher likelihood of live births compared with women with low vitamin D levels (14). Previous studies have demonstrated a relationship between 25OH-D and ovarian reserve parameters anti-mullerian hormone (AMH) and FSH (15). Infertility is defined as the inability of getting pregnant after trying for at least 6 months or one year, for women without use of birth control means assisted reproduction includes all the methods used for fertilization (16). IVF is a technology which continues to evolve. It was developed as a treatment for infertility. The principle of IVF is the retrieval of mature oocytes from a woman’s ovaries, fertilization with sperm, cultivation in the laboratory and transfer of the embryo to the uterine cavity. The first successful treatments in humans were carried out in natural cycles, i.e. relying on a woman’s natural ovulation (17). The chance of a resulting pregnancy with that approach is small. After the development of methods of controlled ovarian hyperstimulation (COH), treatment cycles are at present generally stimulated for retrieval of multiple eggs and thus increased chances of successful treatment due to the possibility of selecting a high quality embryo (s) for transfer (18).

 

MATERIAL AND METHODS:

This study consisted of 60 women undergoing IVF program, aged between 23-42 years with symptoms of PCOS. The subject was recruited from Al-Amal Fertility Center in Baghdad City in the period November 2017 to February 2018. The sonography was performed in the second day of the menstrual cycle. Blood samples were collected on the third day of the menstrual cycle. Clear serum samples were then separated by centrifugation at 4000 rpm for 10 minutes and stored at -20C until use. All serum samples were submitted to AMH determination using Diagnostic Systems Laboratories ELISA kit for AMH. Yhlo-Biotech kit for vitamin D, Boditech Med kit for FSH levels, Estradiol (E2) levels were determined using accubind ELISA microwellskit. There protocol used during ovarian hyperstimulation, is short protocols using GnRH antagonist during the late follicular phase of the stimulation cycle have been utilized. Adding recombinant LH to recombinant FSH protocols, when starting antagonists, as a strategy to increase oocyte yield and improve pregnancy rates. Oocyte retrieval for IVF was then typically scheduled for 30-34 hr thereafter. Then the fertilized oocyte was placed in G1 media for 3-4 days then in G2 media before rewind.

 

Statistical Analysis:

Statistical analysis of data was achieved by (Minitab-version 18.1). The one-way ANOVA test was used for analysis of variance for average hormone level as quantitative variable by qualitative variable. Correlation coefficient (r) between the number of mature oocytes with the different parameters investigated was used. The results in all the above mentioned procedures were accepted as statistically significant when the p-value was less than 5% (p<0.05).


 

Table 1. The mean levels of AMH, E2, FSH and vitamin D in relation to the number of oocytes retrieved upon ovarian stimulation of IVF program.

Parameter

>4 Oocyte

4-8 Oocyte

9-16 Oocyte

<16 Oocyte

F

P

AMH

0.14±0.1

1.164±0.661

2.11±1.286

3.764±2.232

7.31

0.00*

FSH

15.8±0.1

7.22±2.823

8.883±3.129

6.973±2.477

4.62

0.006*

E2

31.00±0.1

22.90±12.77

28.27 ±13.91

34.70±33.38

0.6

0.602

Serum 25(OH)D

13.29±0.1

10.310±2.572

11.115±3.126

9.815±3.087

1.06

0.375

 

Table 2. The mean number of mature and immature oocytes in relation to poor, normal, good and high responders to ovarian stimulation during IVF programs.

Oocytes

>4 Oocyte

4-8 Oocyte

9-16 Oocyte

<16 Oocyte

F

P

Number of mature oocyte

2.00±0.00

5.00±1.581

8.269±2.325

14.583±2.620

51.81

0.000*

Number of immature oocyte

1.00±0.01

2.220±1.202

4.500±1.985

8.583±2.701

24.74

0.000*

 

 

 

Table 3. The mean number of various types of oocytes cases in relation to the different age groups.

Oocyte

≤25 Age (year)

26-36Age (year)

>36 Age (year)

F

P

Number of mature oocyte

12.58±4.73

9.176±3.762

6.714±2.628

7.04

0.002*

Total Number of oocyte

19.21±6.70

14.71±6.06

13.00±8.83

3.61

0.033*

 


RESULTS:

The study populations consisted of 60 females who were undergoing IVF at Al-Amal Fertility Center, in Baghdad. Medical histories showed that the infertility etiology was mostly referred to PCOS. The mean age of the enrolled cases was 26.67±4.26 years. upon stimulation the patients were divided into poor, normal, good and high responders as indicated in the Table 1. The increase in the mean levels of AMH paralleled the increase in the total number of oocytes, showing 0.14±0.1ng/mL, 1.164±0.661ng/mL, 2.11±1.286 ng/mL and 3.764±2.232 ng/mL in poor, normal, good and high responders, respectively. This successive change was found to be significant (F= 7.31 and p=0.000). Similar trend was observed for the mean number of mature oocytes. (F=51.81 and p=0.000) and a significant inverse relationship was found between the Number of mature oocyte with age (F=7.04 and p=0.002). This shows that younger females produce more oocytes. When related to age, IVF outcome showed that the chance of IVF success increased with decreased age (F=4.01 and p=0.01). When the relationship between the number of mature oocytes collected and the investigated parameters was analyzed, only AMH showed positive correlation with the number of mature oocytes (r= 0.527 and p= 0.001). (Table 3).

 

Table 4. Ovarian response of women attending IVF in relation to age

Number of oocytes

%

Age (year)

Poor responders (<4 oocytes) (n=2)

3%

37.00 ± 4.24

Normal responders (4-8 oocytes) (n= 8)

13%

32.13 ± 5.82

Good responders (9-16 oocytes) (n= 28)

46%

29.714 ± 4.845

High responders (> 16 oocytes) (n= 22)

38%

27.05 ± 4.81

F

4.01

P-value

0.011*

 

Table 5. the correlation between the number of mature oocytes collected and the different parameters investigated.

Parameter

r

P

Age

-0.334

0.009*

AMH

0.527

0.001*

FSH

-0.149

0.255

E2

0.183

0.162

25(OH)D

-0.201

0.124

Note: Values are represented with means and ± SD, p*; significant

AMH; Anti-mullerian Hormone, FSH: Follicle Stimulating Hormone, 25(OH)D: 25-hydroxyvitamin D, E2: Estradiol

 

DISCUSSION:

Data presented in this study dealt with 60 females joined in IVF programs. The mean age of the cases in the present study (26.67 years) was close to other studies (19, 20). Females undergoing IVF indicated that ovarian response is better with decreasing age, showing poor response (<4 oocytes) at the oldest age (37.00 years) and high response (>16 oocytes) at the youngest age (27.05years). Consequently, younger females have a better chance of an active IVF. In agreement with this positive trend, who found that the mean serum AMH concentration was more than two and a half-fold in the group with ≥11 collected oocytes as compared to the group with ≤6 oocytes (21). In addition, another study that reported significant increase in good responder women undergoing IVF (≥4 oocytes) as compared to poor responders (<4 oocytes) (19). The current study showed that the mean levels of AMH indicated a progressive increase parallel to the total number of oocytes in poor, normal, good and high responders. Several investigators have reported similar successive increase in the total number of oocytes as a result of increasing AMH levels in responder females undergoing IVF (22,23). The mean number of mature and immature oocytes were significant in different classes of responders to ovarian stimulation from poor to high responders. This finding is in agreement with that found by who reported that the mean number of oocytes was significantly lower in poor respondent women than in normal, good and high respondent women attending IVF programs (21,22). This leads to the conclusion that the ovarian response can be regarded as a reflection of the ovarian reserve. IVF results presented in this study showed that the chance of IVF success increased with decreased age; 28 (46%) of cases were 29.7 years old. The observed high mean levels of AMH can be linked to the increased mean number of collected oocytes. also there was no correlation was observed between vitamin D and the number of mature oocyte that support the suggestion of who suggested that vitamin D deficiency was not associated with the number of follicles and oocytes or with the morphology of the embryo (24), and in a similar study who suggested that vitamin D deficiency had no correlation with IVF outcomes (25). This suggests that AMH levels may be an indicator for ovarian responsiveness, it is worth mentioning here that although AMH level seems to be a good marker for ovarian response to stimulation drug IVF the differences between the mean levels of the remaining parameters including E2 and FSH with total number of oocytes count were not statistically significant among the different classes of responders. The correlation coefficient results indication that, AMH showed positive strong correlation with the number of mature oocytes. This reconfirms the previous result that raised levels of AMH increased the mature number of oocytes, thus offering more chance to higher number of embryos.

 

CONCLUSION:

In conclusion serum AMH levels is significantly decrease with decreasing mature oocytes and it can be the best marker for ovarian reserve and ovarian response in IVF programs and the best marker reflecting the decline of reproductive aging.

 

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Received on 05.01.2019         Modified on 28.02.2019

Accepted on 19.03.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(7): 3527-3530.

DOI: 10.5958/0974-360X.2019.00600.0