Objective: Conclusion: Letrozole and gonadotropin treatments are effective in

Objective: To study the effect of medication (letrozole, and Gonadotropine) on ovulation induction and its
relation with glycoproteins.

Methods: Thirty-three infertile women were enrolled from outpatient women’s
clinic and twenty-two healthy fertile women were enrolled as a control group.
Their ages ranges from (15 to 44) years old. Histidine Rich Glycoprotein (HRG),
Inhibin B and Anti Müllerian hormone (AMH) concentrations were measured in sera
using (ELISA) technique.

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Results: Follow-up examinations four months after receiving treatments revealed
a significant raise in serum HRG concentration (p=0.01) and a considerable decrease
in serum AMH concentration (p=0.04) among infertile women. Pelvic
ultrasonography, showed a significant (p = 0.000) increase in the size and the number
of oocytes in both left and right ovaries infertile women.

Conclusion: Letrozole and gonadotropin treatments are effective in changing the
levels of HRG and AMH in infertile women. 

Keywords: women infertility, HRG, Inhibin B, AMH, letrozole, gonadotropin.

 

Introduction

Infertility is a commonplace ailment that
affects the reproductive system,  described by the lack of ability of couple to
obtain a clinical pregnancy after one year or more of
regular unprotected sexual intercourse 1.

Due
to wars in Iraq, around 2003, the Iraqi environment suffered from acts of profanation.
 Large numbers of injuries and deaths
were caused by the destructive chemicals and radioactive materials. The people
who survived these devastating incidences either suffered from cancer or
infertility. However, there are only a few studies that have examined the
postwar examined specifically on infertility 2.

In this study, we aim to address the effect of
letrozole and gonadotropin hormones for stimulating ovulation in infertile females.
Serum glycoproteins (HRG, Inhibin B, and AMH) will be measured.

 

           Histidine-rich glycoprotein (HRG) is a
multi-domain protein concerned in the coagulation, angiogenesis and immune response,
all of which are importance for institution of a pregnancy 3.

         Inhibins
are dimeric polypeptide hormones that belong to the transforming growth factor
? (TGF-?) super family. Inhibin B, a paracrine ovarian and testicular regulator
is secreted by the granulosa cells of the ovary has multiple paracrine effects
on the utero-placental unit, instead of promising marker poverty for both male
and female 4.                                                                  

      
   Anti-Müllerian hormone (AMH) is
a glycoprotein released by the granulosa cells of preantral and
small antral follicles and participates in folliculogenesis instruction 9. It
has a main role in cell growth and differentiation 5.  

          Gonadotropins are hormones (luteinizing
hormone (LH) and follicle-stimulating hormone (FSH)) given to stimulate a
woman’s ovaries to make follicles, which contain oocytes 6.

       
Letrozole can prevent the transform of androgens to estrogen, this lead to release the
hypothalamic-pituitary axis from the negative feedback of estrogen, produces a
rise of FSH secretion from the anterior pituitary 7.

 

Materials and Methods

     This study included thirty-three infertile
women; enrolled from the outpatient women’s clinic in the period from October
2016 to April 2017. Their age’s ranged between (15-44 years old). The control
group was consisted of 22 healthy fertile women subjects who have not signs and symptoms of diseases and whose ages were
matched with the age of infertile women. The practical part was carried out in
AL-Hussein Teaching Hospital of Kerbala. A questionnaire was documented to
obtain the information on female fertility. The first blood samples were drawn on
the second day of menses from both infertile and control females to measure the
glycoproteins (HRG), Inhibin B and (AMH) levels. After that, the infertile
women have been given medication, letrozole plus gonadotropin for (four months),
and the blood samples were treatments.

Concentrations of the glycoproteins in this study were
determined by enzyme-linked immune sorbent assay (ELISA) system (Bio Tek
Instruments 21 7337, U.S.A).
Pelvic ultrasonography was used to measure the size and number of oocytes.

 

Statistical Analysis                                                                                                    

The results are expressed as Mean ± Standard Error
(SE). Student t-test and Pearson’s correlation coefficients was used to analyze
results by using Statistical Package for the Social Sciences (SPSS) version
22.0. P-value ? 0.05 was considered significant 8.

 

 

Results and Discussions

The clinical characteristics of infertile women                                  

The present study includes 33 infertile women and 22 control
fertile women. The number of infertile women, age, BMI, family history, residency,
education, and type of infertility, regulation of menses, abortion history, and
causes of infertility were documented in (Table 1).

Table (1) the clinical characteristics    feature of    infertile women

 

(%)                     

No. = 33   

Variables                

72.72
18.18
9.09

24
6
3

Age(years)
15  –  29
30  –  40
41 –   45

36.36
42.42
21.21

12
14
7

BMI(Kgm) :
Normal   
(18.5-24.9 )
Over weight ( 25-29.9)
Obese  (    > 35 
)

30.30
69.69

10
23

Family history :
With family
Without family

72.72
27.27

24
9

Residency :
City
Urban

63.63
36.36

21
12

Education :
No Educated
Educated

39.39
60.60

13
20

Type of infertility
Primary
Secondary

48.48
51.51

16
17

Regulation of menses
Regular
I rregular

30.30
69.69

10
23

Abortion:
With abortion
Don’t abortion

21.21
12.12
66.6

7
4
22

Causes of infertility:
Tubal factor
Endometriosis
Unexplained

 Measurement
of the glycoproteins in infertile women and control groups

            Comparing the serum levels of glycoproteins HRG, inhibin
B and AMH between infertile and healthy subjects: the results revealed that
infertile women have a significantly lower serum inhibin B level than the healthy women (p = 0.05), whereas there was no any
significant differences in the
concentration of their HRG and AMH levels (p > 0.05) (Table
2).

Table (2): The concentrations of HRG, Inhibin B and
AMH of infertile and control groups     

P-value

Control               
No.=22             Mean ± SE

Infertile women          No.= 33  

Mean ± SE

Parameters         
        

0.08

48.46  ± 
2.21

24.87  ± 
7.17  

HRG ( ng/ml)       

0.05

319.34
±  6.55

241.9
±  20.43

Inhibin B ( pg/ml)

0.06

3.70  ±  0.48

5.30  ±  0.68  

AMH  ( ng/ml)     

 

Follow up of infertile women

       The
treatment group of 33 infertile women were followed up for four months after the
treatment with medication drug (Gonadotrophin and Letrozole), and then the
parameters under study were measured.

Measurement of glycoprotein (HRG, Inhibin B, AMH)

The result showed a significant decrease in AMH concentration (p = 0.04)
and a significant increase in HRG concentration (p= 0.01) among infertile women
whereas there was no significant differences change in inhibin B level (p >
0.05) (Table 3).

 

Table (3):
The concentrations of the glycoproteins (HRG, Inhibin B and AMH) of infertile   women before and after treatment     

 
P-value

   After treatment   
  
No. = 33
Mean ± SE        

Before
 treatment
No. = 33      
Mean ± SE           

 
Parameters                 

0.01

57.96±10.77

24.89 
±   7.17

HRG  ( ng/ml)

0.14

283.77±19.70

242.00 
±  20.43

InhibinB (pg/ml) 

0.04

3.87± 0.49

5.55 
±  0.65

AMH    (ng/ml)

 

            Pelvic ultrasonography technique was used
to measure the follicle sizes after ovary stimulation in the treatment group.
The results revealed a significant (p=0.000) increase in size of follicles
to became mature oocytes in both left and right ovary of infertile women after the treatment compared (Table 4).

 
P-value

After
treatment
     No. = 33
    Mean ± SE

Before  treatment
   No. = 33
 Mean ± SE

 

0.000

20.03±0.53

8.015 
±  0.21

Size of
oocyte

0.000

     2.54 
±  0.15

19.15
±1.11

No. of
oocyte

 Table (4): The size and number of oocytes
of  infertile women before and after treatment

 

Discussion

        
Inhibin B does seem concerning to fertility, as low levels of inhibin B is
related to impaired ovulation, low pregnancy rates and increased risk of
miscarriage 9.  Groome, et al. (1994)
indicate that inhibin B a granulosa cell product has a main role in follicular
growth with the possibility that serum inhibin B level correlates with follicular
function and oocyte number 10.

     
  Another study by Klein, et al.
(1996) indicated that decline inhibin B secretion was a reflection of a reduced ovarian follicular pool in older women.  Magoffin and Jakimiuk, (1997)  found that the amount of both Inhibin proteins
secreted into follicular fluid seems to increase with follicle development,
although their concentrations may faintly decrease in the largest follicles due
to strength in a greater fluid volume 11. 
Inhibin B is an important indicator of ovarian reserve (the ovary’s
capacity to respond to gonadotropin stimulation), predicts magnitude of
retrievals, and is used to determine Ovarian Hyper-stimulation Syndrome
determines gonadotropin dosage for Assisted Reproductive Technologies (ART) 12.    Chang,
et al. (2002) found that inhibin B in follicular fluid may serve as an
effective marker for follicular development 13.

Histidine-rich glycoprotein interacts with other
angiogenic factors, such as vascular endothelial growth factor VEGF and
fibroblast growth factor FGF but no studies to date have shown how HRG affects
angiogenesis in the follicle 14. Apparently, the exact role of HRG in
reproduction remains to be investigated as its exact bimolecular function is
unclear 15.

         
In this study, although there was no significant difference in the
concentration of AMH, but there was increase in the level of AMH in infertile
women group comparing with control group. The latest studies have shown that
AMH consider a good predictor of ovarian reserve and the success rates of in
vitro fertilization IVF though; both AMH and FSH are still used as ovarian reserve test 16.
Takahashi, et al. (2008) showed that there was no significant
correlation between AMH levels and oocyte number 17.

          Gonadotropin therapy plays an essential
role in ovarian stimulation for infertility management.  In the last century, efforts have been made
over to improve gonadotropin preparations. Undoubtedly, current gonadotropins
have best quality and safety as well as clinical activity than previously
ones.  A major performance has been
introducing recombinant technology in the manufacturing processes for
follicle-stimulating hormone, luteinizing hormone, and human chorionic
gonadotropin 18. Gonadotropins play a significant role in the secretion of
several substances by granulosa cells (eg: hyaluronic acid) in turn affecting
oocyte development and maturation 19 as well as treatment of infertility 20.
Gonadotrophin therapy is based on the physiological concept that initiation and
maintenance of follicle growth may be achieved by a transient raise in FSH
above a threshold dose for sufficient duration to generate a limited number of
developing follicle 21.

       
The studied of HRG genotype affects the number of fertilized oocytes.
Women given lower gonadotropins dosages get the most fertilized oocytes. The
number percentage did not differ based on HRG genotype. This indicates that the
HRG does not appear to influence oocyte maturity 22.

        The level of AMH gradually depleted after gonadotropin treatment during controlled ovarian
stimulation. This decline could be an effective directly or indirectly
on the negative influence of FSH on AMH ovarian secretion.                                                                                                 

         The exogenous
FSH medication elevates estradiol, which could be the source of AMH drooping as estradiol negatively influences the regulation of
AMH in the ovary, so it is recommended to use initial low doses of
gonadotropins and protocols with a GnRH antagonist. It is also necessary to
direct women to fertility therapy facilities as soon as possible to select the right
treatment 23. Recently, it has been suggested that AMH may exhibit a
physiological role in down regulating the aromatizing capacity of granulosa
cells until the time of follicular selection 24. 

           Letrozole
has become a significant drug in our armamentarium for treating infertility, yet surprisingly
little effort has been devoted toward optimizing its effectiveness 25. Letrozol
as an aromatase inhibitor, when using it in the initial follicular phase has a
negative feedback effect on the hypothalamus and pituitary glands lead to GnRH,
LH and FSH secretion with resultant ovarian follicular growth stimulation. letrozole
and its drug group has safety, reliable and cheap with therapeutic effect. It
is likely that letrozole does not produce harmful effects similar to that found
with clomiphene citrate on the endometrium, even
though it can cause pregnancy 26.  Durlinger, et al. (2002) demonstrated
that AMH and inhibin B are produced by granulosa cells and appear to have both
autocrine and paracrine effects within the follicle. In larger, pre-antral
follicles, the oocyte increases in size as the follicle develops more layers of
granulosa cells.  The later stages of
follicle maturation from antrum formation to ovulation are gonadotrophin:
dependent and are highly regulated by the cyclical changes in LH and FSH.
During these gonadotrophin dependent stages, the primary oocyte does not grow
significantly but undergoes both cytoplasmic and nuclear maturation 27.

      

Conclusion

·        
Inhibin B is
considered a better marker for predicting infertility than AMH.

·        
HRG and AMH are responsive to  
leterozol and gonadotrophin treatment, as there is an increase in the
level of HRG and decrease in the level of AMH in infertile women group after
treatment.

The
antral follicle developed into mature oocyte after medication with
gonadotrophin plus letrozole.