Objective: To study the effect of medication (letrozole, and Gonadotropine) on ovulation induction and itsrelation with glycoproteins. Methods: Thirty-three infertile women were enrolled from outpatient women’sclinic 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 serausing (ELISA) technique.Results: Follow-up examinations four months after receiving treatments revealeda significant raise in serum HRG concentration (p=0.01) and a considerable decreasein serum AMH concentration (p=0.04) among infertile women. Pelvicultrasonography, showed a significant (p = 0.000) increase in the size and the numberof oocytes in both left and right ovaries infertile women. Conclusion: Letrozole and gonadotropin treatments are effective in changing thelevels of HRG and AMH in infertile women.
Keywords: women infertility, HRG, Inhibin B, AMH, letrozole, gonadotropin. IntroductionInfertility is a commonplace ailment thataffects the reproductive system, described by the lack of ability of couple toobtain a clinical pregnancy after one year or more ofregular unprotected sexual intercourse 1.Dueto wars in Iraq, around 2003, the Iraqi environment suffered from acts of profanation. Large numbers of injuries and deathswere caused by the destructive chemicals and radioactive materials. The peoplewho survived these devastating incidences either suffered from cancer orinfertility. However, there are only a few studies that have examined thepostwar examined specifically on infertility 2.In this study, we aim to address the effect ofletrozole and gonadotropin hormones for stimulating ovulation in infertile females.Serum glycoproteins (HRG, Inhibin B, and AMH) will be measured.
Histidine-rich glycoprotein (HRG) is amulti-domain protein concerned in the coagulation, angiogenesis and immune response,all of which are importance for institution of a pregnancy 3. Inhibinsare dimeric polypeptide hormones that belong to the transforming growth factor? (TGF-?) super family. Inhibin B, a paracrine ovarian and testicular regulatoris secreted by the granulosa cells of the ovary has multiple paracrine effectson the utero-placental unit, instead of promising marker poverty for both maleand female 4. Anti-Müllerian hormone (AMH) isa glycoprotein released by the granulosa cells of preantral andsmall antral follicles and participates in folliculogenesis instruction 9. Ithas a main role in cell growth and differentiation 5. Gonadotropins are hormones (luteinizinghormone (LH) and follicle-stimulating hormone (FSH)) given to stimulate awoman’s ovaries to make follicles, which contain oocytes 6. Letrozole can prevent the transform of androgens to estrogen, this lead to release thehypothalamic-pituitary axis from the negative feedback of estrogen, produces arise of FSH secretion from the anterior pituitary 7. Materials and Methods This study included thirty-three infertilewomen; enrolled from the outpatient women’s clinic in the period from October2016 to April 2017.
Their age’s ranged between (15-44 years old). The controlgroup was consisted of 22 healthy fertile women subjects who have not signs and symptoms of diseases and whose ages werematched with the age of infertile women. The practical part was carried out inAL-Hussein Teaching Hospital of Kerbala. A questionnaire was documented toobtain the information on female fertility.
The first blood samples were drawn onthe second day of menses from both infertile and control females to measure theglycoproteins (HRG), Inhibin B and (AMH) levels. After that, the infertilewomen have been given medication, letrozole plus gonadotropin for (four months),and the blood samples were treatments. Concentrations of the glycoproteins in this study weredetermined by enzyme-linked immune sorbent assay (ELISA) system (Bio TekInstruments 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 analyzeresults by using Statistical Package for the Social Sciences (SPSS) version22.
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 controlfertile women.
The number of infertile women, age, BMI, family history, residency,education, and type of infertility, regulation of menses, abortion history, andcauses 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 Measurementof the glycoproteins in infertile women and control groups Comparing the serum levels of glycoproteins HRG, inhibinB and AMH between infertile and healthy subjects: the results revealed thatinfertile women have a significantly lower serum inhibin B level than the healthy women (p = 0.05), whereas there was no anysignificant differences in theconcentration of their HRG and AMH levels (p > 0.05) (Table2).Table (2): The concentrations of HRG, Inhibin B andAMH 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 Thetreatment group of 33 infertile women were followed up for four months after thetreatment with medication drug (Gonadotrophin and Letrozole), and then theparameters 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 womenwhereas 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 usedto measure the follicle sizes after ovary stimulation in the treatment group.The results revealed a significant (p=0.
000) increase in size of folliclesto 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 oocytesof infertile women before and after treatment Discussion Inhibin B does seem concerning to fertility, as low levels of inhibin B isrelated to impaired ovulation, low pregnancy rates and increased risk ofmiscarriage 9. Groome, et al. (1994)indicate that inhibin B a granulosa cell product has a main role in folliculargrowth with the possibility that serum inhibin B level correlates with follicularfunction 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 proteinssecreted into follicular fluid seems to increase with follicle development,although their concentrations may faintly decrease in the largest follicles dueto strength in a greater fluid volume 11. Inhibin B is an important indicator of ovarian reserve (the ovary’scapacity to respond to gonadotropin stimulation), predicts magnitude ofretrievals, and is used to determine Ovarian Hyper-stimulation Syndromedetermines gonadotropin dosage for Assisted Reproductive Technologies (ART) 12. Chang,et al. (2002) found that inhibin B in follicular fluid may serve as aneffective marker for follicular development 13.Histidine-rich glycoprotein interacts with otherangiogenic factors, such as vascular endothelial growth factor VEGF andfibroblast growth factor FGF but no studies to date have shown how HRG affectsangiogenesis in the follicle 14.
Apparently, the exact role of HRG inreproduction remains to be investigated as its exact bimolecular function isunclear 15. In this study, although there was no significant difference in theconcentration of AMH, but there was increase in the level of AMH in infertilewomen group comparing with control group. The latest studies have shown thatAMH consider a good predictor of ovarian reserve and the success rates of invitro fertilization IVF though; both AMH and FSH are still used as ovarian reserve test 16.Takahashi, et al. (2008) showed that there was no significantcorrelation between AMH levels and oocyte number 17. Gonadotropin therapy plays an essentialrole in ovarian stimulation for infertility management. In the last century, efforts have been madeover to improve gonadotropin preparations.
Undoubtedly, current gonadotropinshave best quality and safety as well as clinical activity than previouslyones. A major performance has beenintroducing recombinant technology in the manufacturing processes forfollicle-stimulating hormone, luteinizing hormone, and human chorionicgonadotropin 18. Gonadotropins play a significant role in the secretion ofseveral substances by granulosa cells (eg: hyaluronic acid) in turn affectingoocyte development and maturation 19 as well as treatment of infertility 20.Gonadotrophin therapy is based on the physiological concept that initiation andmaintenance of follicle growth may be achieved by a transient raise in FSHabove a threshold dose for sufficient duration to generate a limited number ofdeveloping follicle 21. The studied of HRG genotype affects the number of fertilized oocytes.
Women given lower gonadotropins dosages get the most fertilized oocytes. Thenumber percentage did not differ based on HRG genotype. This indicates that theHRG does not appear to influence oocyte maturity 22. The level of AMH gradually depleted after gonadotropin treatment during controlled ovarianstimulation. This decline could be an effective directly or indirectlyon the negative influence of FSH on AMH ovarian secretion. The exogenousFSH medication elevates estradiol, which could be the source of AMH drooping as estradiol negatively influences the regulation ofAMH in the ovary, so it is recommended to use initial low doses ofgonadotropins and protocols with a GnRH antagonist. It is also necessary todirect women to fertility therapy facilities as soon as possible to select the righttreatment 23.
Recently, it has been suggested that AMH may exhibit aphysiological role in down regulating the aromatizing capacity of granulosacells until the time of follicular selection 24. Letrozolehas become a significant drug in our armamentarium for treating infertility, yet surprisinglylittle effort has been devoted toward optimizing its effectiveness 25. Letrozolas an aromatase inhibitor, when using it in the initial follicular phase has anegative feedback effect on the hypothalamus and pituitary glands lead to GnRH,LH and FSH secretion with resultant ovarian follicular growth stimulation. letrozoleand its drug group has safety, reliable and cheap with therapeutic effect.
Itis likely that letrozole does not produce harmful effects similar to that foundwith clomiphene citrate on the endometrium, eventhough it can cause pregnancy 26. Durlinger, et al. (2002) demonstratedthat AMH and inhibin B are produced by granulosa cells and appear to have bothautocrine and paracrine effects within the follicle. In larger, pre-antralfollicles, the oocyte increases in size as the follicle develops more layers ofgranulosa cells. The later stages offollicle 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 growsignificantly but undergoes both cytoplasmic and nuclear maturation 27. Conclusion· Inhibin B isconsidered a better marker for predicting infertility than AMH.· HRG and AMH are responsive to leterozol and gonadotrophin treatment, as there is an increase in thelevel of HRG and decrease in the level of AMH in infertile women group aftertreatment.Theantral follicle developed into mature oocyte after medication withgonadotrophin plus letrozole.