Download prolactin and other hormonal profiles in sudanese infertile females

PROLACTIN AND OTHER HORMONAL
PROFILES IN SUDANESE INFERTILE
FEMALES
By:
Inaam Mohammed Elhassan Satti Fadul
B. Sc (Science and Education)
Faculty of Education
University of Khartoum
Supervisor:
Dr. Asia Mohammed El Hassan
A thesis submitted in the partial fulfillment of the requirements for the
degree of Master Biochemistry
Faculty of Veterinary Medicine
University of Khartoum
May 2006
Table of contents
Table of contents …….…………………………………….….....................i
Dedication ………………………………...…………….…………….…. v i
Acknowledgments ………………………………….……….. ……….…v ii
Abstract English ……………………………………….…………......... v iii
Abstract Arabic …………………………………………………………….x
List of figures …..………………………………….……………… …....xii
List of tables ………………………………………….………………… xiv
Introduction ....…………………………………………………….......... xv
Chapter one
Literature Review
1.1
Prolactin ………………………………...…………..………….…1
1.1.1
Prolactin structure …………………………………………….…1
1.1.2. Physiologic Effect of Prolactin ………..……….…...……….…..2
1.1.3. Regulation of Prolactin Secretion ………….…..……….……....3
1.2.
Hyperprolactinemia …………..……...……………………….....6
1.2.1 Consequences of hyperprolactinemia ………………………….6
1.2.2. Causes of hyperprolactinemia …………..………..………….….8
1.2.2.1 Tumors of the pituitary gland ……………………………….... 8
1.2.2.2. Thyroid gland disorder:…………………………………….…..8
1.2.2.3. Drugs:……………………………………………...……………..9
1.2.2.4. Macroprolactinemia …………………………………………….9
i
1.2.3. Hyperprolactinemia in female infertility …………………….…9
1.2.4. Hyperprolactinemia in Polycystic Ovary Syndrome ………….10
1.2.5 Psychological distress in patients with hyperprolactinemia ..... 10
1.2.6. Hyperprolactinemia and bone mineral density ……..……...….11
1.3. Anovulation ………………………………………………………..11
1.4. Gonadotrophic hormones ………………………...…………….…11
1.4.1. Gonadotropin structure …………………………….……….…..12
1.4.2. Gonadotropin functions ………………………………………...12
1.4.2.1. Luteinizing Hormone ……………………………...…….…….12
1.4.2.2. Follicle-Stimulating Hormone ……………………………..….13
1.4.3. Control of Gonadotropin secretion :……………………….…...13
1.5. Estrogens …………………………………………………………...15
1.5.1. Secretion of Estrogens ………………………………………….. 15
1.5.2. Biologic actions of estrogens …………………………...………..15
1.6. The menstrual cycle ……………………………………..………...16
1.7. Relationship of prolactin and gonadotrophic hormones in
regulation of the ovarian function …..………………………………...18
Chapter two
MATRIALS AND METHODS
2.1. Materials …………………………………………………………...20
2.1.1. Study population …………………………………………..…….20
ii
2.1.1.1. Human subjects for control experiments ………….………..20
2.1.1.2. Infertile female subjects ……………………………...………..20
2.1.2. Samples . ………………………………………………………….20
2.1.3. Equipment ……………………………………………...…….…..21
2.1.4. Reagents …………………………………………………..……..21
2.1.4.1 Reagents for prolactin estimation ………………...…………..21
2.1.4.2. Reagent for Follicle Stimulating Hormone (FSH) estimation.21
2.1.4.3. Reagents for Luteinising Hormone (LH) estimation ……….22
2.1.4.4. Reagents for Estradiol (E2) estimation ……………...……….22
2.2. Methods ………………………………………………………….…22
2.2.1. Prolactin estimation ……………………………………………..22
2.2.1.1. Prolactin EIA Assay Procedure ……………………………....22
2.2.2. Follicle stimulating Hormone estimation ……………………....25
2.2.2.1. FSH EIA Assay Procedure …………………………………...25
2.2.3. Luteinising Hormone estimation ………………………………25
2.2.3.1. LH EIA Assay Procedure …………………………………....25
2.2.4. Estradiol estimation ………………………………………….…25
2.2.5 Survey ……………………………………………………………29
2.2.6. Study duration and area ………………………………………..29
2.2.7. Statistical analysis Procedure ………………………………….29
iii
Chapter three
Result
3.1. Measurement of serum hormones …………………………….…..30
3.1.1 Serum prolactin levels …………………………………………....30
3.1.2. Serum gonadotropin levels ………………………………………33
3.1.3 Comparing gonadotropin with PRL levels ……………………...37
3.1.4. Menstrual cycle pattern in hyperprolactinemia ………………..38
3.1.5 LH/FSH ratio ……………………………………………………...41
3.1.6. Serum estradiol levels ……………………………………………41
3.1.7. Measurement of esradiol (E2), PRL , FSH , and LH in infertile
patients …………………………………………………………………..41
3.2 Questionnaire data analysis ………………………………………..44
3.2.1 Age ………………………………………………………………....44
3.2.2 Environmental and genetical factors ……………………………48
3.2.3. Pathologic factors ……………………………………………….52
3.3 Incidence and prevalence of hyperprolactinemia in Sudanese female
patients infertility ………………………………………………………56
Chapter four
Discussion
4.1 Female infertility …………………………………………………...57
iv
4.1.1. Infertile patients with high levels of serum prolactin ………...57
4.2 Hyperprolactinemia and menstrual patterns …………………….59
4.3 Serum estradiol levels ……………………………………………...62
4.4. Questionnaire data analysis ……………………………………...62
4.4.1. Age ………………………………………………………………..62
4.4.2. Geographical and ethnic distribution ………………………….63
4.4.3. Environmental and genetical factors …………………………..63
4.4.4. Associated Patholigies …………………………………………..64
4.4.5 The incidence of hyperprolactinemia in Sudanese females … 65
conclusions ……………………………………………………………. 66
recommendations ……………………………………………..……… 66
References……………………………………………………………....68
Appendix ……………………………………………………………….80
v
To:
The Soul of my father with great love.
My mother whose dedication, live and continuous
support
remained an unlimited source of encouragement for
me.
My brother and sisters who did their best to make
the environment around me conductive and healthy
allowing this effort to materialize throughout the
investigations and writing
With respect
vi
ACKNOWLEDGEMENTS
I pray thankfully to Allah whose will permitted the completion of this
work.
I am greatly indebted to my supervisor Dr. Asia Mohamed El Hassan for
her valuable advice, guidance, continuous encouragement and patience
throughout my study.
I am especially grateful to Prof. Abdel Latiff Ashmaig the chairman of
Reproductive Health Care Centre for his assistance by allowing me to do the
analysis of the samples in their centre.
Thanks are extended to Dr. Mohamed Siddig and Mr. Hassan for their
help during the laboratory investigations.
Sincere thanks are due to my brother Abdel Moneim and my family for
their support and encouragement.
vii
ABSTRACT
Background and Objectives: Infertility is a distressing problem for couples
seeking to have children. Hyperprolactinemia is one of the causes of female
infertility which in itself is caused by a variety of conditions and factors.
The objective was to investigate hyperprolactinemia in infertile Sudanese
females and how other related hormones namely gonadotropins and estrogen
interact in such a condition. The study also aimed to throw some light on the
interplay of environmental, genetical and physiological factors.
Materials and Methods: Seventy infertile hyperprolactinemic females
patients were selected from 385 patients visiting the Reproductive Health
Care Centre in Khartum. The investigations were carried out to estimate
prolactin (PRL), follicle stimulating hormone (FSH), luetinizing hormone
(LH) and estradiol (E2). High sensitive and highly specific enzyme immunoassay techniques were used for hormone determinations.
A structured questionnaire was designed to shed light on the interplay and or
the effect of environmental, genetical and some pathological conditions with
respect to hyperprolactinemia and associated infertlty.
Results: Infertile females were found to have high levels of prolactin ranging
between 600- 1800 mIU/L and accordingly placed into three categories.
These were Group 1(G1) with moderately high (670.07 + 10.4), Group2
(G2) with high (959.0 + 27.5) and Group3 (G3) with very high (1679.7 +
95.1) prolactin levels, all of which were significantly high when compared to
the control (146.93 + 21.6); P=0.001 for G1and G2 and P=0.0001 G3. The
mean values of FSH(G1 12.81 ± 0.8, G2 11 ± 0.16, G3 8 ± 0.14) and LH (G1
viii
8.92 ± 1.09, G2 8.45 ± 0.98, G3 6.5 ± 0.87)of the three groups were
compared with mean values of FSH( 14.15 ± 1 ) and LH( 10.33 ± 0.45 ) in
the control group. A consistent drop, in the levels of both FSH and LH with
increasing degree of hyperprolactinemia is observed. The drop is not
significant for infertile females in G1 with moderately high prolactin level.
However, it is significant (P< .01) for G2 and G3 whose hyperprolactinemia
is almost equal to or over 1000 mlIU/L.
Checking the estrogen level (2.2433 + 0.06) in these patients showed a
highly significant (P< 0.001) reduction in its levels when compared to
control (29.2 + 3.0). Investigation of menstrual cycle patterns in
hyperprolactinemic
patients
revealed
three
classes:
patients
with
regular(RC), irregular cycles (IC) and those who suffered amenorrhea (AM).
The amenorrheaic patients showed the highest levels of PRL (1412.6 + 9.7)
when compared to control (146.93 ± 21.6) and their gonadotroins (FSH
=18.36 + 2.8, LH =16.60 + 2.0) were significantly increased compared to
controls (FSH 14.15 ± 1, LH 10.33 ± 0.7 )
Questionnaire data analysis showed that hyperprolactinemic patients were
mostly from Central (Sinar, White Nile and Gezira region + khartoum)
Sudan; 57% and Northern Sudan were 23%.
From close relative incidences it was found that 25% of the patients had
close relatives with infertility problems. 8.57% of the hyperprolactinemic
infertile females suffered hypothyroidism and were on medication to treat
hypothyrodism, 2.86% were hypertensive using antihypertensive drugs and
no diabetic cases.
Discussion and Conclusion: It is concluded that the problem of
hyperprolactinemia is one of the causes of infertility in Sudan. The condition
ix
is a very complex one that can be caused and affected by hormonal,
environmental and genetical factors.
x
‫ﻣﻠﺨﺺ ﺍﻷﻃﺮﻭﺣﺔ‬
‫ﺍﳌﻘﺪﻣﺔ ﻭﺍﻻﻫﺪﺍﻑ‪ :‬ﻋﺪﻡ ﺍﳋﺼﻮﺑﺔ ﻣﺸﻜﻠﺔ ﻣﺆﺭﻗﺔ ﻟﻸﺯﻭﺍﺝ ﺍﻟﺬﻳﻦ ﻳﺴﻌﻮﻥ ﻟﻺﳒﺎﺏ ‪ ،‬ﻓﺮﻁ ﺇﻓﺮﺍﺯ ﺑﺮﻭﻻﻛﺘﲔ‬
‫ﺍﻟﺪﻡ ﻫﻮ ﺃﺣﺪ ﻣﺴﺒﺒﺎﺕ ﻋﺪﻡ ﺍﳋﺼﻮﺑﺔ ﻫﺬﻩ ﻟﺪﻯ ﺍﻹﻧﺎﺙ ‪ .‬ﻭ ﻫﻮ ﻧﻔﺴﻪ ﺗﺴﺒﺒﻪ ﻇﺮﻭﻑ ﻭ ﻋﻮﺍﻣﻞ ﻣﺘﻨﻮﻋﺔ‪.‬‬
‫ﻭ ﻗﺪ ﻛﺎﻥ ﺍﳍﺪﻑ ﻫﻮ ﲝﺚ ﻭ ﺩﺭﺍﺳﺔ ﺣﺎﻻﺕ ﻧﺴﺎﺀ ﺳﻮﺩﺍﻧﻴﺎﺕ ﻣﺼﺎﺑﺎﺕ ﺑﻌﺪﻡ ﺍﳋﺼﻮﺑﺔ ‪ ،‬ﻭ ﻣﻌﺮﻓﺔ ﻛﻴﻔﻴـﺔ‬
‫ﺗﻔﺎﻋﻞ ﺑﻌﺾ ﺍﳍﺮﻣﻮﻧﺎﺕ ﺍﻷﺧﺮﻯ ﺫﺍﺕ ﺍﻟﻌﻼﻗﺔ ﻛﺎﻟﻔﻮﻧﺎﺩﻭﺗﺮﻭﺑﲔ ﻭ ﺍﻹﺳﺘﺮﻭﺟﲔ ﰲ ﻫﺬﻩ ﺍﳊﺎﻟﺔ‪.‬‬
‫ﻛﻤﺎ ﻫﺪﻓﺖ ﺍﻟﺪﺭﺍﺳﺔ ﺇﱃ ﺗﺴﻠﻴﻂ ﺑﻌﺾ ﺍﻟﻀﻮﺀ ﻋﻠﻰ ﺗﻀﺎﻓﺮ ﺍﻟﻌﻮﺍﻣﻞ ﺍﻟﺒﻴﺌﻴـﺔ ﻭ ﺍﻟﻮﺭﺍﺛﻴـﺔ ﻭ ﺍﻟﻔـﺴﻴﻮﻟﻮﺟﻴﺔ‬
‫ﻭﺍﳌﺮﺿﻴﺔ‪.‬‬
‫ﺍﳌﻮﺍﺩ ﻭﺍﻟﻨﻈﺮﻳﺎﺕ‪ :‬ﰲ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﰎ ﺇﺧﻀﺎﻉ ﻋﺪﺩ ﺳﺒﻌﲔ ﻣﻦ ﺍﻟﻨﺴﺎﺀ ﺍﻟﻼﺋﻲ ﻳﻌﺎﻧﲔ ﻣﻦ ﻋﺪﻡ ﺍﳋﺼﻮﺑﺔ ﻣﻊ‬
‫ﻓﺮﻁ ﺇﻓﺮﺍﺯ ﺍﻟﱪﻭﻻﻛﺘﲔ ﻟﻠﺪﺭﺍﺳﺔ ﺍﺧﺘﺮﻥ ﻣﻦ ﺑﲔ ‪ 385‬ﻣﺮﻳﻀﺔ ﺯﺭﻥ ﺍﳌﺮﻛﺰ ﺍﻻﺳﺘﺸﺎﺭﻱ ﻟـﺼﺤﺔ ﺍﻷﻣﻮﻣـﺔ‬
‫ﻭﺍﻟﻄﻔﻮﻟﺔ ﻭﺍﻃﻔﺎﻝ ﺍﻻﻧﺎﺑﻴﺐ‬
‫ﻟﻘﺪ ﰎ ﲢﺪﻳﺪ ﻫﺮﻣﻮﻧﺎﺕ ﺍﻟﱪﻭﻻﻛﺘﲔ ﻭ ﺍﳍﺮﻣﻮﻧﺎﺕ ﺍﻟﺘﻜﺎﺛﺮﻳﺔ ‪ ،‬ﺍﳍﺮﻣﻮﻥ ﺍﳊﻮﺻﻠﻲ ﺍﳌﻨﺒﻪ )‪ (FSH‬ﻭ ﻫﺮﻣﻮﻥ‬
‫ﺍﳉﺴﻢ ﺍﻷﺻﻔﺮ )‪ (LH‬ﻭ ﺍﻹﺳﺘﺮﺍﺩﻳﻮﻝ ﰲ ﺍﻟﻨﺴﺎﺀ ﺍﳌﺼﺎﺑﺎﺕ ﺑﻌﺪﻡ ﺍﳋﺼﻮﺑﺔ ﺑﺎﺳـﺘﺨﺪﺍﻡ ﺃﺳـﻠﻮﺏ ﻋـﺎﱄ‬
‫ﺍﳊﺴﺎﺳﻴﺔ ﻭ ﺍﻟﺘﺨﺼﻴﺺ ﻟﻠﻤﻘﺎﻳﺴﺔ ﺍﳌﻨﺎﻋﻴﺔ ﻟﻺﻧﺰﳝﺎﺕ‪.‬‬
‫ﺍﻟﻨﺘﺎﺋﺞ‪ :‬ﻭﺿﻌﺖ ﺍﻹﻧﺎﺙ ﺍﳌﺼﺎﺑﺎﺕ ﺑﻌﺪﻡ ﺍﳋﺼﻮﺑﺔ ﻭ ﺍﻟﻼﺋﻲ ﻳﻌﺎﻧﲔ ﻣﻦ ﻓﺮﻁ ﺇﻓﺮﺍﺯ ﻫﺮﻣﻮﻥ ﺍﻟـﱪﻭﻻﻛﺘﲔ ﰲ‬
‫ﺍﻟﺪﻡ ﰲ ﳎﻤﻮﻋﺎﺕ ﺛﻼﺙ ﺣﺴﺐ ﻣﺴﺘﻮﻳﺎﺕ ﺍﻟﱪﻭﻻﻛﺘﲔ ‪ :‬ﻣﻌﺘـﺪﻝ ﺍﻻﺭﺗﻔـﺎﻉ )‪ (10.4 ± 670.07‬ﻭ‬
‫ﻋﺎﱄ ﺍﻻﺭﺗﻔﺎﻉ )‪ (27.5 ± 959.0‬ﻭ ﻋﺎﱄ ﺍﻻﺭﺗﻔﺎﻉ ﺟﺪﺍ ً)‪ (95.1 ± 1679.7‬ﻣﻘﺎﺭﻧـﺔ ﲟﺠﻤﻮﻋـﺔ‬
‫ﺍﻟﺘﺤﻜﻢ )‪ .(21.6 ± 146.92‬ﻭ ﰲ ﻛﻞ ﺍ‪‬ﻤﻮﻋﺎﺕ ﻛﺎﻧﺖ ﻣﺴﺘﻮﻳﺎﺕ ﻛ ﱟﻞ ﻣﻦ ﺍﳍﺮﻣﻮﻥ ﺍﳊﻮﺻﻠﻲ ﺍﳌﻨﺒﻪ ﻭ‬
‫ﻫﺮﻣﻮﻥ ﺍﳉﺴﻢ ﺍﻷﺻﻔﺮ ﻣﻨﺨﻔﻀﺔ ﺑﺼﻮﺭﺓ ﻣﻠﺤﻮﻇـﺔ ﺍﳍﺮﻣـﻮﻥ ﺍﳊﻮﺻـﻠﻲ ﺍﳌﻨﺒـﻪ ﰲ ﺍ‪‬ﻤﻮﻋـﺔ ﺍﻻﻭﱃ‬
‫)‪ (0.87 ± 12.81‬ﻭﺍ‪‬ﻤﻮﻋﺔ ﺍﻟﺜﺎﻧﻴﺔ )‪ (0.16 ± 11‬ﻭﺍ‪‬ﻤﻮﻋﺔ ﺍﻟﺜﺎﻟﺜﺔ )‪ . (0.14 ± 8‬ﻫﺮﻣﻮﻥ ﺍﳉـﺴﻢ‬
‫ﺍﻻﺻﻔﺮ ﰲ ﺍ‪‬ﻤﻮﻋﺔ ﺍﻻﻭﱃ )‪ (1.09 ± 8.92‬ﻭﺍ‪‬ﻤﻮﻋﺔ ﺍﻟﺜﺎﻧﻴـﺔ )‪ (0.98 ± 8.45‬ﻭﺍ‪‬ﻤﻮﻋـﺔ ﺍﻟﺜﺎﻟﺜـﺔ‬
‫)‪ (0.87 ± 6.5‬ﻣﻘﺎﺭﻧ ﹰﺔ ﲟﺠﻤﻮﻋﺎﺕ ﺍﻟﺘﺤﻜﻢ ﺍﻟﱵ ﻛﺎﻧﺖ )‪ (1 ± 14.15‬ﻟﻠـﻬﺮﻣﻮﻥ ﺍﳊﻮﺻـﻠﻲ ﺍﳌﻨﺒـﻪ‬
‫ﻭ)‪ (0.45 ± 10.33‬ﳍﺮﻣﻮﻥ ﺍﳉﺴﻢ ﺍﻻﺻﻔﺮ ‪.‬‬
‫ﻋﻨﺪ ﻣﺮﺍﺟﻌﺔ ﻣﺴﺘﻮﻯ ﺍﻻﺳﺘﺮﻭﺟﲔ ﻟﺪﻯ ﺑﻌﻀﻬﻦ ‪ ،‬ﻭﺟﺪ ﻫﺒﻮﻁ ﻭﺍﺿﺢ ﰲ ﻣﺴﺘﻮﻳﺎﺗﻪ )‪. (0.6 ± 2.2433‬‬
‫ﻣﻘﺎﺭﻧﺔ ﲟﺠﻤﻮﻋﺎﺕ ﺍﻟﺘﺤﻜﻢ )‪. (0.3 ± 29.20‬‬
‫ﰎ ﻓﺤﺺ ﺍﻟﻨﺴﺎﺀ ﺍﻟﻼﺋﻲ ﻳﻌﺎﻧﲔ ﻣﻦ ﻋﺪﻡ ﺍﳋﺼﻮﺑﺔ ﻛﺬﻟﻚ ﻟﻠﻜﺸﻒ ﻋﻦ ﻛﻴﻔﻴﺔ ﺗﺄﺛﲑ ﻓﺮﻁ ﺇﻓﺮﺍﺯ ﺑـﺮﻭﻻﻛﺘﲔ‬
‫ﺍﻟﺪﻡ ﰲ ﺃﻧﻈﻤﺔ ﺩﻭﺭﺍ‪‬ﻦ ﺍﻟﺸﻬﺮﻳﺔ ‪ ،‬ﻭﻭﻓﻘﹰﺎ ﻟﺬﻟﻚ ﻓﻘﺪ ﰎ ﻭﺿﻌﻬﻦ ﰲ ﳎﻤﻮﻋﺎﺕ ﺛﻼﺙ‪ :‬ﳎﻤﻮﻋـﺔ ﻣﻨﺘﻈﻤـﺔ‬
‫‪xi‬‬
‫ﺍﻟﺪﻭﺭﺓ ﻭ ﳎﻤﻮﻋﺔ ﻏﲑ ﻣﻨﺘﻈﻤﺔ ﺍﻟﺪﻭﺭﺓ ﻭ ﳎﻤﻮﻋﺔ ﻣﻨﻘﻄﻌﺔ ﻋﻨﻬﺎ ﺍﻟﺪﻭﺭﺓ ‪ .‬ﺃﻇﻬﺮﺕ ﳎﻤﻮﻋﺔ ﺍﻧﻘﻄﺎﻉ ﺍﻟـﺪﻭﺭﺓ‬
‫ﺃﻋﻠﻰ ﻣﺴﺘﻮﻳﺎﺕ ﺍﻟﱪﻭﻻﻛﺘﲔ )‪ (9.7 ± 1412.63‬ﻣﻘﺎﺭﻧﺔ ﲟﺠﻤﻮﻋﺎﺕ ﺍﻟﺘﺤﻜﻢ )‪.(21.6 ± 146.93‬‬
‫ﻭﻛﺎﻧﺖ ﻫﺮﻣﻮﻧﺎﺕ ﺍﻟﻘﻮﻧﺎﺩﻭﺗﺮﻭﺑﲔ ﻟﺪﻳﻬﻦ ﺯﺍﺋﺪﺓ ﺑﺼﻔﺔ ﻣﻠﺤﻮﻇﺔ ﺍﳍﺮﻣﻮﻥ ﺍﳊﻮﺻﻠﻲ ﺍﳌﻨﺒﻪ )‪(2.8 ± 18.36‬‬
‫ﻭﻫﺮﻣﻮﻥ ﺍﳉﺴﻢ ﺍﻻﺻﻔﺮ )‪ (2 ± 16.6‬ﻣﻘﺎﺭﻧـﺔ ﲟﺠﻤﻮﻋـﺎﺕ ﺍﻟـﺘﺤﻜﻢ ﻟﻠـﻬﺮﻣﻮﻥ ﺍﳊﻮﺻـﻠﻲ ﺍﳌﻨﺒـﻪ‬
‫)‪ (1 ± 14.15‬ﻭﳍﺮﻣﻮﻥ ﺍﳉﺴﻢ ﺍﻻﺻﻔﺮ )‪(0.7 ± 1.33‬‬
‫ﻭ ﻗﺪ ﺃﻇﻬﺮ ﲢﻠﻴﻞ ﺑﻴﺎﻧﺎﺕ ﺍﻻﺳﺘﺒﻴﺎﻥ ﺃﻥ ﺍﻟﻨﺴﺎﺀ ﺍﻟﻼﺋﻲ ﻳﻌﺎﻧﲔ ﻓﺮﻁ ﺇﻓﺮﺍﺯ ﺑﺮﻭﻻﻛﺘﲔ ﺍﻟﺪﻡ ﻛﻦ ﻣـﻦ ﻭﺳـﻂ‬
‫ﺍﻟﺴﻮﺩﺍﻥ )ﺳﻨﺎﺭ‪ -‬ﺍﻟﻨﻴﻞ ﺍﻻﺑﻴﺾ‪ -‬ﺍﳉﺰﻳﺮﺓ( ﻭﺍﳋﺮﻃﻮﻡ )‪ ، (%57‬ﺍﻟﻌﺎﻣﻞ ﺍﻟﻮﺭﺍﺛﻲ ﻛﺎﻥ ﻣﻦ ﺑﲔ ﻣﺴﺒﺒﺎﺕ ﻓﺮﻁ‬
‫ﺇﻓﺮﺍﺯ ﺑﺮﻭﻻﻛﺘﲔ ﺍﻟﺪﻡ ‪ .%25‬ﰲ ﻳﻌﺾ ﺍﳌﺮﻳﻀﺎﺕ ﻳﺒﺪﻭ ﺃﻥ ﺍﺳﺘﺨﺪﺍﻡ ﻋﻘﺎﻗﲑ ﺍﺭﺗﻔﺎﻉ ﺿﻐﻂ ﺍﻟﺪﻡ ﻭ ﻣﻌﺎﳉـﺔ‬
‫ﺍﳌﺼﺎﺑﺎﺕ ﺑﺎﻟﻐﺪﺓ ﺍﻟﺪﺭﻗﻴﺔ ﻗﺪ ﺗﻜﻮﻥ ﺃﺣﺪ ﺍﳌﺴﺒﺒﺎﺕ‪.‬‬
‫ﺍﳋﻼﺻﺔ‪ :‬ﻗﺪ ﺗﻮﺻﻠﺖ ﺍﻟﺪﺭﺍﺳﺔ ﺇﱃ ﺃﻥ ﻣﺸﻜﻠﺔ ﻋﺪﻡ ﺍﻻﺧﺼﺎﺏ ﻣﻌﻘﺪﺓ ﻭﺗﺴﺒﺒﻬﺎ ﻋﺪﺓ ﻋﻮﺍﻣﻞ ﻣﻨـﻬﺎ ﺗـﺄﺛﲑ‬
‫ﺍﳍﺮﻣﻮﻧﺎﺕ ﻭﺍﻟﺒﻴﺌﺔ ﻭﺍﻟﻌﻮﺍﻣﻞ ﺍﻟﻮﺭﺍﺛﻴﺔ ‪.‬‬
‫‪xii‬‬
List of figures
Page No
Fig (1): Preovulatory follicle ………………………………………….………... 13
Fig (2) : Illustrates the feedback loops that controls the secretion
of LH and FSH……………………………………………………………………..14
Fig 3 : approximate plasma concentrations of the gonadotropins
and ovarian hormones during the normal female ………………………….. 17
Fig. (4): Prolactin levels in infertile patients and control ………………. 32
Fig. (5): FSH levels in infertile patients and control …………………….35
Fig. (6): LH levels in infertile patients and control …………..………… 36
Fig. ( 7): Control Prolactin, FSH and LH Levels in Infertile
Patients …………………………………………………………………..37
Fig. 8 Menstrual cycle pattern …………………………………..………40
Fig. (9): Prolactin and estradoil …………………………………………42
Fig (10): LH and estradoil ………………………………………………43
Fig. (11): FSH and estradoil …………………………………………….44
Fig.(12): Percent age groups of the patients ……………………………46
Fig13 Marriage duration ……………………………………………….. 47
Fig. (14): Residential regions of the patients …………………………...49
Fig.(15): Area of residence during the last 5 years ……………………..50
Fig. (16): Family history of infertility …………………………………..51
xiii
List of figures
Page No
Fig (17): Drugs used by hyperprolactinemic infertile females ………….52
Fig. (18) Thyroid diseases ………………………………………………53
Fig.(19) Hypertensive patients ………………………………………….54
Fig. (20): Diabetic patients ……………………………………………...55
xiv
List of tables :
List of tables
Page No.
Table (1): Grouping of infertile female based on PRL levels …………..31
Table (2):
Prolactin and gonadotropin in the three groups of infertile
females ………………………………………………………………….34
Table (3) Levels of PRL, FSH and LH in patients with different menstrual
cycle patterns: ……………………………………….………………..…39
Table (4) Estradiol, prolactin and gonadotropins in hyperprolacinemic
infertile females: ……………………………………………...…………41
xv
Introduction
Throughout the world, millions of couples suffer involuntarily from
infertility, and this causes a variety of personal and social problems.
Infertility in general, would appear to affect 5 – 15 % of couples of the
reproductive age group of the word’s population. This percentage represents
35 – 105 million couples of which 10 – 20 % will be infertile without a
cause being identified (WHO. 1984).
Infertility is defined as the inability to conceive after more than one year of
unprotected intercourse (WHO. 1975). It is considered as primary when
there is no history of a pervious pregnancy and secondary when the female
has previously become pregnant, but cannot achieve a new gestation
(Acosta, and Nieves, 1988).
Traditionally in past times, the female has been considered as the prime
cause of barren marriages. However, over time the concept of a "male
factor" has emerged, and now male infertility is widely recognized.
According to current data, about one third of cases of infertility are
attributable to male factors, one third to female factors, and one third to both
male and female factors (Baum 1988).
Data from the world fertility surveys suggest that the percentage of infertile
couples as well as the prevalence of its different causes varies from one
country to another and among different countries (WHO. 1984). The
variation in the levels and patterns of infertility and the relationship between
them depends on the interplay between "core" fertility that includes
chromosomal, congenital, and hormonal abnormalities, and "acquired"
infertility due to infections as well as environmental and occupational
xvi
factors (Mohamed 1992). Among these hormonal abnormalities is primarily
targeted for purposes of diagnosis and management. Hormonal abnormalities
include an array of irregularities of endocrine functions of the pituitary and
the gonads affecting levels of follicle stimulating hormone (FSH),
leutinizing hormone (LH), prolactin, estrogen and progesterone. Other
hormonal anamolies may involve the thyroid gland.
objectives:
The basic objectives of this study will be the following:1- To in evaluate the significance and prevalence of
hyperprolactinemia in Sudanese female patient’s infertility.
2- To investigate the relationship between prolactin, FSH, LH
and oestradiol.
3- To assess the predominant cause and look into curability of
the disorder.
4- To study the menstrual patterns in hyperprolactinic infertility
Sudanese females.
xvii
Chapter one
Literature Review
1.1. Prolactin:
Prolactin (PRL) is a polypeptide hormone secreted by the anterior pituitary gland,
whose main role is to stimulate lactation in the postpartum period. Increases in
prolactin secretion can be physiological (pregnancy and lactation) or pathological
(hypothalamic and pituitary disease). The suppression of the hypothalamicpituitary-gonadal axis and the resistance of the ovary to gonadotropin action are
induced in hyperprolactinemia resulting in amenorrhea and lack of ovulation.
Infertility associated with hyperprolactinemia in females can be reversed with
treatment. A recent progress in female sterility reported that the ovulatory
disturbances occupy the second position of the female sterile factor (Loneseu et
al. 2002 ). The precise endocrinological examination must be done for the
diagnosis of the ovulatory disturbances. Generally clomiphene citrate is used for
moderate hypothalamic anovulations, and bromocriptine is for prolactin related
diseases and endocrinological polycyctic ovary (Aisake 1997).
1.1.1. Prolactin structure:
Human prolactin consists of 199 amino acids and has three intramolecular
disulfide bonds. Prolactin circulates in the blood predominantly in a monomeric
form (23 kd) but it also exists in a dimeric (48 to 56 kd) and polymeric (> 100 kd)
forms (Wilson et al. 1998). Although the major form of prolactin found in the
pituitary gland is 23 kd, variants of prolactin have been characterized in many
mammals, including humans. Prolactin variants can be results of alternative
splicing of the primary transcript, proteolytic cleavage and other posttranslational
modifications of the amino acid chain.
1
1.1.2. Physiologic Effect of Prolactin:
Prolactin, the major hormone in lactogenesis, action is modulated by a
combination of second messenger activities that include cyclic nucleotides,
prostaglandin, calcium ion changes, polyamine production and growth factors. In
pregnancy cAMP and cGMP increase progressively and may be involved in
stimulation of the mitogenic and morphogenic processes that occur with
pregnancy. At delivery cAMP levels falls precipitously, and cGMP levels
continue to rise and stay elevated in the lactation period (Peterson 1997).
Prolactin is best known for the multiple effects it exerts on the mammary gland.
The varied effects of prolactin on the mammary gland include growth and
development of the mammary gland (mammogenesis), synthesis of milk
(lactogenesis), and maintenance of milk secretion (galactopoiesis). Although it
has long been accepted that prolactin is involved in the development of the
mammary gland (Bern and Nicoll 1968), recent elegant techniques have
confirmed such findings. Targeted disruption of the prolactin gene (prolactin
knockout) (Horseman et al.1997) or knockout of the prolactin receptor (Ormandy
et al., 1997) results in abnormal mammogenesis characterized by complete
absence of lobuloalveolar units in adult homozygous females. Prolactin knockout
heterozygotes
appear
to have
nearly
normal
mammogenesis
that
is
indistinguishable from wild type (Horseman et a.l 1997).
However, it also exerts effects on other targets important to the reproduction of
the mammalian species. In some mammals, particularly rodents, prolactin is also
important for the maintenance and secretory activity of the corpus luteum. It also
affects other actions related to reproduction such as mating and maternal
behaviors.
2
Lactogenesis clearly requires pituitary prolactin, since hypophysectomy during
pregnancy prevents subsequent lactation (Nelson and Gaunt 1936). In the process
of lactogenesis, prolactin stimulates uptake of some amino acids, the synthesis of
the milk proteins casein and -lactalbumin, uptake of glucose, and synthesis of the
milk sugar lactose as well as milk fats (Bar ber et al., 1992; Tuker 1994)
Actions of prolactin on luteal function depend on species and the stage of the
estrous cycle. In rodents, prolactin can either be luteotrophic after mating or
luteolytic in the absence of a mating stimulus.
In most rodents, prolactin acts as a luteotrophic hormone by maintaining the
structural and functional integrity of the corpus luteum for 6 days after mating
(Morishige and Rothchild 1974). This "luteotrophic" action of prolactin, which
has been best described in the rat, is characterized by enhanced progesterone
secretion . Progesterone is essential for the implantation of the fertilized ovum
(along with estrogen), maintenance of pregnancy, and inhibition of ovulation
(Freeman 1994).
Prolactin enhances progesterone secretion two ways: prolactin potentiates the
steroidogenic effects of luteinizing hormone (LH) in granulosa-luteal cells
(Richards and Williams 1967) and inhibits the 20-hydroxysteroid dehydrogenase
enzyme, which inactivates progesterone (Freeman 1994).
1.1.3. Regulation of Prolactin Secretion:
Prolactin, like all anterior pituitary hormones is secreted in an episodic
manner((Wilson 1998). Plasma concentrations of prolactin are the highest during
sleep and the lowest during the waking hours in humans (Parker et al., 1974;
Sassin et al., 1973). Recent human volunteer experiments proved, however, that
this rhythm of prolactin secretion is maintained in a constant environment
3
independent of the rhythm of sleep (Waldstreisher 1996) although with
considerably larger amplitude in women than men. The best-known physiological
stimulus affecting prolactin secretion is the suckling stimulus applied by the
nursing young. Cessation of the suckling stimulus results in termination of
prolactin secretion, and the rate of decrease in blood prolactin levels is
proportional to the metabolic clearance rate of the hormone (Grosvenor et al.,
1979 ;Nagy and Halasz 1983) Moreover, the amount of prolactin released is
related to the intensity of the stimulus. In humans (Diaz et al., 1989 ; Stern and
Rechlin 1990) and cattle (Lefcourt et al.,1994) the prolactin-secretory response to
nursing is superimposed by the endogenous circadian rhythm of prolactin
secretion; that is, the same intensity of suckling stimulus can elevate prolactin
levels more effectively at certain times of day when the circadian input enhances
the suckling stimulus-evoked secretory response.
Hypothalamic control of the preovulatory proestrous surge of prolactin is far
from clear. Although it is clear that dopamine inhibits prolactin secretion and that
removal of the tonic dopaminergic inhibition results in enhanced prolactin
secretion, the role of dopamine as a regulator of the proestrous surge of prolactin
is contradictory.
Its secretion is enhanced by various prolactin releasing factors (Wilson et al.,
1998).
It is clear that prolactin secretion is dramatically affected by "stress." A myriad of
stresses have been used to characterize such effects on prolactin secretion. These
include, but are not limited to, the following: ether stress (Bank et al., 1994;
Grattan and Waverill1991; Johnston and Negro-Vilar1986; Kaji et al., 1985;
Lookingland, et al., 1990; Muri and Ben-Jonathan 1987; Neill 1970; (Yogev et
al., 1994; restraint stress Demarest et al., 1984; Gala and Haisenlder 1986;
4
Jurcovicova et al., 1990; Kehoe et al., 1991; Ramalho et al.,1995; thermal stress
Vaha-Eskeli et al., 1991; hemorrhage Burns and Sarkar 1993; Jurcovicova et al.,
1990; social conflict Haisenleder et al., 1986) and even academic stress in
humans (Malarkey et al., 1991).
Because, in most cases, the prolactin-secretory response (i.e., stimulation or
inhibition) differs depending on the nature of stress, one cannot describe a unitary
mechanism but must define the mechanism associated with each specific stress
modality.
The most important physiological stimuli that elevate pituitary prolactin secretion
are suckling (Neill 1974; Terkel et al.1972; stress Neill 1970 and increased levels
of ovarian steroids, primarily estrogen Neill 1970; Nill e ta,l 1982). Such stimuli
are transduced by the hypothalamus which elaborates a host of PRF and
prolactin-inhibiting factors (PIF) (Fig. 2). In mammals, the control exerted by the
hypothalamus over pituitary prolactin secretion is largely inhibitory (BenJonathan1985). On the other hand, the hypothalamus is also involved in the acute
stimulatory control of prolactin secretion by removal of the inhibition
(disinhibition) and/or superimposition of brief stimulatory input. In addition,
prolactin secretion is also influenced by numerous factors released by the
lactotrophs themselves (autocrine regulation) or by other cells within the pituitary
gland (paracrine regulation).
5
1.2. Hyperprolactinemia:
Hyperprolactinemia means the presence of abnormally high levels of prolactin in
the circulation(Haslett et al.,2002) Normal levels are less than 580 mIU/L for
women, and less than 450 mIU/L for men. It is the most common clinical
hypothalamic-hypophysis
disorder.
Sexual dysfunction in women with
hyperprolactinemia is not uncommon. A significant percent of women with
evaluated hyperprolactinemia had sexual dysfunction (Kadioglu et al.,2005).
European investigators have measured prolactin levels in females presenting with
abnormalities of the menstrual cycle and have reported a 15-30 % incidence of
hyperprolactinemia (Seppala, et al.,1975). Their data confirm this finding and
suggest that many women suffering from oligomenrrhea or secondary
amenorrhea may suffer from a disorder of prolactin secretion.
1.2.1. Consequences of hyperprolactinemia:
An excess of prolactin can suppress ovulation and can be symptomatic of
hypothyroidism or luteal phase defects. In women, symptoms of this condition
include galactorrhea (the production of breast milk by non-nursing women) and
anovulation (when a woman does not ovulate).
Of greater concern, however, is that this abnormality of prolactin is associated
with an absence of cyclic gonadotropin secretion (Tyeson, et al.,1948) resulting
in menses cessation, amenorrhea which can be primary or secondary.
Primary amenorrhea can be diagnosed if a patient has normal secondary sexual
characteristics but no menarche by 16 years of age. If a patient has no
secondary sexual characteristics and no menarche, primary amenorrhea can be
diagnosed as early as 14 years of age. Secondary amenorrhea is the absence of
6
menses for three months in women with previously normal menstruation and
for nine months in women with previous oligomenorrhea. Secondary
amenorrhea is more common than primary amenorrhea (The Practice
Committee of the American Society For Reproductive Medicine 2004;
Speroff,L. and Fritz 2005).
Although amenorrhea and anovulation are the most usual clinical findings but
other alterations of gonadal function as oligomenorrhea or luteal phase alterations
can be found. Galactorrhea appears in approximately 30 % of patients, but its
presence in women with ovulation disorders is highly suggestive of
hyperprolactinemia (Rosaro and Garofalo 2002). The action of PRL in supraphysiological levels on the ovaries or on the hypothalamic-pituitary axis for the
release of gonadotropins leads to inhibition of the cyclic functioning of the
pituitary gland and of the ovaries. The consequences are either the production of
immature follicles marked by anovulatory or dysovulatory cycles, or absence of
follicle production marked by amenorrhea. Thus, prolactin plays a major role in
the reproductive system not only by its lactotropic but also by its
antigonadotropic effects (Bessioud, et al.2002). The relationship between
elevated prolactin levels and menstrual cycle in infertile Sudanese females was
studied and the results showed that 36 % of regularly menstruating infertile
Sudanese were hyperprolactinaemic. The incidence of hyperprolactinemia among
oligomenorrhoeic group was reported to be 20% (5).
When menarche has failed to occur or menstrual cycle have stopped, the problem
can be traced back to a functional or structural defect in the hypothalamus,
pituitary, ovaries, or uterus (Norrison 1998).
7
Subjects with primary amenorrhea and delayed puberty can present with
hyperprolactinemia (Rosaro and Garofalo, 2002). Primary amenorrhea caused by
hyperprolactinemia is a rare condition. Bromocriptine therapy, however, proved
useful in inducing ovulation and cause the menarche onset (Kawano.et al.,2000).
Increased prolactin levels will lead to decreased bone density when compared
with healthy individuals of the same age. The longer someone has
hyperprolactinemia, the lower the bone mass will become. The more abnormal
menstrual function is, the lower the bone density (Hergenroeder 1995).
1.2.2 Causes of hyperprolactinemia:
Hyperprolactinemia can be caused by several factors such as:
1.2.2.1 Tumors of the pituitary gland:
Prolactinomas are the most common pituitary tumors (Hattori 2003; Biller 1999).
Prolactinomas represent a 60 % of pituitary tumors with various symptoms,
hormonal and reproductive abnormalities. It has been pointed that prolactinomas
prevailed among females in reproductive age with a higher incidence of
macroadenoma. The most common alterations related to reproductive tract seen
were menstrual irregularities, galactorrhea, infertility, hyperprolactinemia,
hypogonadism and abnormalities of puberty (Hurtado et al.,,2004).
1.2.2.2. Thyroid gland disorder:
The hyperprolactinemia of hypothyroidism is related to several mechanisms. In
response to the hypothyroid state, a compensatory increase in the discharge of
central hypothalamic thyrotropin-releasing hormone results in increased
8
stimulation of prolactin secretion. Furthermore, prolactin elimination from the
systemic circulation is reduced, which contributes to increased prolactin
concentrations.(Asa and Ezzat 2002).
1.2.2.3. Drugs:
Drugs that block the effects of dopamine at the pituitary or deplete dopamine
stores in the brain may cause the pituitary to secrete prolactin. These drugs
include the major tranquilizers, high blood pressure medications, and antinausea
drugs. Oral contraceptives and recreational drugs (such as marijuana)
1.2.2.4. Macroprolactinemia:
Patients with macroprolactinaemia are clinically characterized by the lack of
amenorrhea and galactorrhea ie asymptomatic with intact gonadal and
reproductive function and pregnancy was possible despite a marked
hyperprolactinemia (Hattori 2003).
This term should not be confused with
macroprolactinoma, which refers to a large pituitary tumour greater than 10 mm
in diameter. Macroprolactinemia refers to a polymeric form of prolactin in which
several
prolactin
molecules
form
a
polymer
that
is
recognized by
immunologically based serum assays. In general, macroprolactin results from the
binding of prolactin to IgG antibodies. The large prolactin polymer is unable to
interact with the prolactin receptor. Little, if any, biological effect of prolactin
excess is noted(Vallette-Kasic et al., 2002). It was found that anti-PRL
autoantibody is the leading cause of macroprolactinemia that might be
heterogeneous in nature.
1.2.3. Hyperprolactinemia in female infertility:
A prospective study was undertaken to determine the incidence of
hyperprolactinemia in a group of referred infertile females. Of 133 referred
9
infertility patients, 19.5% had elevated levels of serum prolactin. 4.4% patients
with hyperprolactinemia had neither abnormal menstrual function nor
galactorrhea. Hyperprolactinemia is a common finding in an infertile population,
more so when galactorrhea and / or menstrual dysfunction is also present
(Kredentser et al.,1981).
1.2.4. Hyperprolactinemia in Polycystic Ovary Syndrome:
The polycystic ovary (PCO) results from a systemic hormonal dysfunction
(Gonzalez et al., 2003). With the use of multiple blood sampling, the prevalence
of hyperprolactinemia, in 150 patients with polycystic ovary syndrome was found
to be 17% (Luciano et al., 1984). The clinical features of PCO may include
chronic anovulation, hyperandrogenism, and bilaterally enlarged multicytic
ovaries (Goldzieher 1981). The endocrine profile of patients with (PCO) reflects
the mild elevations in serum or urinary androgens and normal to elevated serum
lutenizing hormone (LH) with suppressed follicle stimulating hormone (FSH)
levels, resulting in an increased LH / FSH ratio (Yen.1980). It was concluded that
a population with PCO has a higher risk of infertility, anovulation, obesity,
hyperandrogenism, and abnormal menses.
1.2.5. Psychological distress in patients with hyperprolactinemia:
It is now recognized that prolactin has effects on behavior. Both prolactin and its
receptor are found within various regions of the brain (Harlan et al.,,1983;
Bridges et al.,,1935; Emanuele et al., 1992 and Dutt et al.,1994). In clinical
settings it has been observed that patients with hyperprolactinemia may display
psychological symptoms (Fava et al.,1981). It has been found that
hyperprolactinemic patient reported significant increase in symptom of
depression, anxiety and hostility (Reavley et al.,.1997).
10
1.2.6. Hyperprolactinemia and bone mineral density:
Studies in women with hyperprolactinemia resulting from pituitary tumors have
demonstrated high rates of osteoporosis believed to result from hypoestrogenism.
Preliminary surveys have indicated that schizophrenia patients also may have
elevated rates of osteoporosis and pathological fractures, possibly resulting in
part from the long-term administration of antipsychotic agent that produce
hyperprolactinemia and secondarily low estrogen and testosterone levels. This
potential complication of treatment with certain antipsychotic agents requires
careful study and could represent a serious public health problem as the
schizophrenia prevalence is about 10% worldwide. Such patients are at increased
risk for osteoporosis because of several reasons including poor diet, lack of
exercise and excessive smoking (Naildoo et al., 2003).
1.3. Anovulation:
Anovulation, a common cause of female infertility, is a curable condition
(Homburg 2003). The disorder of ovulation accounts for about 30 % of infertility
and often presents with irregular periods or absence of menses associated with
hyperprolactinemia. (Fairley and Taylor 2003).
1.4. Gonadotrophic hormones:
These are two hormones secreted from cells in the anterior pituitary called
gonadotrophs. Most gonadotrophs secrete only Luteinizing hormone LH also
called luteotropin or Follicle Stimulating hormone FSH, also called
folliculotropin but some appear to secrete both hormones.
11
1.4.1. Gonadotropin structure:
As thyroid-simulating hormone, LH and FSH are large glycoproteins composed
of alpha and beta subunits. The alpha subunit is identical in all three of these
anterior pituitary hormones, while the beta subunit is unique and endows each
hormone with the ability to bind its own receptor.
1.4.2. Gonadotropin functions:
FSH is the hormone that stimulates the growth and secretion of ovarian follicle in
females. LH is responsible for stimulation in females and the conversion of the
ovulated ovarian follicle into an endocrine structure called a corpus luteum (Fox
2002).
1.4.2.1. Luteinizing Hormone
In both sexes, LH stimulates secretion of sex steroids from the gonads. In the
testes, LH binds to receptors on Leydig cells, stimulating synthesis and secretion
of testosterone. In females LH is responsible for stimulation of theca cells in the
ovary to secrete testosterone, which is converted into estrogen by adjacent
granulosa cells. Consequently this stimulates conversion of the ovulated ovarian
follicle into an endocrine structure called a corpus luteum (Demarest et al.,1984).
which secrete the steroid hormones progesterone. Progesterone is necessary for
maintenance of pregnancy, and, in most mammals, LH is required for continued
development and function of corpora lutea. The name luteinizing hormone
derives from this effect of inducing luteinization of ovarian follicles.
12
1.4.2.2. Follicle-Stimulating Hormone
As its name implies, FSH stimulates
the maturation of ovarian follicles.
Administration of FSH to
humans and animals induces
Fig (1): Preovulatory follicle
"superovulation", or development of more than the usual number of mature
follicles and hence, an increased number of mature gametes Fig(1). FSH is also
critical for sperm production. It supports the function of Sertoli cells, which in
turn support many aspects of sperm cell maturation.
1.4.3. Control of Gonadotropin secretion :
The principle regulator of LH and FSH secretion is gonadotropin-releasing
hormone (GnRH) also known as LH-releasing hormone. GnRH is a ten amino
acid peptide that is synthesized and secreted from hypothalamic neurons and
binds to receptors on gonadotrophs.
As depicted in the figure(2) below, GnRH stimulates secretion of LH, which in
turn stimulates gonadal secretion of the sex steroids testosterone, estrogen and
progesterone. In a classical negative feedback loop, sex steroids inhibit secretion
of GnRH and also appear to have direct negative effects on gonadotrophs.
This regulatory loop leads to pulsatile secretion of LH and, to a much lesser
extent, FSH. The number of pulses of GnRH and LH varies from a few per day to
one or more per hour. In females, pulse frequency is clearly related to stage of the
cycle. Numerous hormones influence GnRH secretion, and positive and negative
control over GnRH and gonadotropin secretion is actually considerably more
13
complex than depicted in the figure. For example, the gonads secrete at least two
additional hormones; inhibin and activin which selectively inhibit and activate
FSH secretion from the pituitary
Fig (2) : Illustrates the feedback loops that controls the secretion of LH and
FSH.
14
1.5. Estrogens:
Three compounds with estrogen activity have been isolated. All are C18 steroids.
β-Estradiol is the hormone normally secreted by the ovarian follicle. Estrone and
estriol are largely products of estradiol metabolism. The ovary is the principal
site of estrogen production in the nonpregnant female. In females estrogens are
also products of the fetal-placental unit.
1.5.1. Secretion of Estrogens:
In adult women during the reproductive years the daily secretion of estrogens
varies cyclically during the menstrual cycle. Daily secretions of estradiol or
estrone are 0.1 to 0.2 mg / day at time of ovulation in mid cycle. Estrogen
secretion is determined by the rate of estrogen synthesis, which is governed by
the two pituitary gonadotropins LH and FSH.
1.5.2. Biologic actions of estrogens:
(a) Reproductive system:Although estrogens affect nearly all vertebrate tissues to some degree, their
principal effect is stimulation of growth and maturation of organs composing the
female’s reproductive system and the maintance of its reproductive capacity.
These include the uterus, vagina, breasts and also influence pattern of pubic and
axillary hair. In addition there may be local effects exerted within the ovary to
stimulate growth of follicles, a process normally controlled by the pituitary
gonadotropins.
(b) Bone metabolism:
That estrogens regulate normal bone metabolism is suggested from the bone
decalcification seen in postmenopausal osteoporosis in the human female.
Estrogens promote closer of bony epiphyses similar to that of androgens, so that
girls deficient in estrogens prior to completion of puberty may demonstrate a lack
of epiphseal closure and grow abnormally tall (Smith et al.,,1983).
15
1.6. The menstrual cycle:
The normal reproductive years of a female are characterized by monthly
rhythmical changes in the rates of secretion of the female hormones and
corresponding changes in the sexual organs themselves. This rhythm is called the
female sexual cycle or menstrual cycle. The duration of the cycle averages 28
days, and may be as short as 20 days or as long as 45 days even in completely
normal females. Abnormal cycle length is often associated with decreased
fertility. However, the two significant results of the female sexual cycle are:
1- Only a single ovum is normally released from the ovaries each month,
so that normally only a single fetus can grow at a time.
2- The uterine endometrum is prepared for implantation of the fertilized
ovum at required time of the month.
The ovarian changes during the sexual cycle are completely dependant on
gonadotropic hormones secreted by the anterior pituitary. Ovaries that are not
stimulated by these gonadtropic hormones remain inactive, which is the case
throughout childhood when almost no gonadotropic hormones are secreted.
However begins secreting proressivly more and more gandotropic hormones,
which culminates in the initiation of monthy sexual cycle between the age 11 and
15 years; this culmination is called menarche. The anterior pituitary secretes two
different hormones that are known to be essential for function of the ovaries FSH
and LH during each month of the female sexual cycle, there is a cyclic increase
and decrease of them as illustrated in figure (3), (Gugton and Hall1997).
16
(From Gyton and Hall 1997)
17
Diagnosis and management of abnormal menstrual function must be based
upon an understanding of the regulation of the normal cycle. Dynamic
relationships exist between the hypothalamic, pituitary and gonadal
hormones which allow for the cyclic nature of the normal female
reproductive process. These hormonal changes are correlated with
morphologic changes in the ovary, making the coordination of this system
one of the most remarkable event in biology (Speroff et al.,.1983).
The ovarian cycle takes place as a result of the interaction of the central
nervous system (CNS) and the hypothalamo – hypophysial – ovarian axis.
The CNS and the hypothalamus constitute the control for the coordinating
elements which in turn act as signal generators. The hypophysic converts the
hypothalamic signals to gonado0trophins and the ovaries guarantee the
cyclical control through feedback mechanism. Alterations in any of the
normal steps will lead to in fertility as manifested by an ovulation, in
adequate ovulation or the production of an in sufficient corpus lutcum. The
disturbance in ovarian function is
usually, but not always, manifested by an abnormality in the pattern of
menstruation, e.g. amenorrhea, oligomenorrhea or some variability in
menstrual cycle length (Baird 1979; De Acosta and Andino1985).
1.7. Relationship of prolactin and gonadotrophic hormones in
regulation of the ovarian function:
Circulating concentration of the LH, FSH, prolactin (PRL), oestrdiol- 17
beta (E2) were instigated in female with normal cycle and proven fertility,
with irregular cycles and in fertility and with primary or secondary
amenorrhea in order to understand the relationship of the prolactin and
gonadotrophic hormones in regulation of the ovarian function. The results
indicate that hyperprolactinemia inhibits the pituitary to release the mid
18
cycle LH surge in response to positive feedback action of the elevated
oestrogen at the mid cycle. The elevated LH and FSH level found in the
cases of the secondary amenorrhea is the indication of the absence of the
steroid negative feedback due to the ovarian failure (Khana et al., 1990).
19
CHAPTER TWO
MATRIALS AND METHODS
2.1. Materials:
2.1.1. Study population:
2.1.1.1. Human subjects for control experiments
Fifteen females of proven fertility and in their reproductive age, who were
not suffering from any systemic endocrine disease, were selected to serve as
a control group, their age range was 15 – 45 years.
2.1.1.2. Infertile female subjects:
a)
Three hundred and eighty five infertile females with a medical
history of primary or secondary infertility were studied. Their ages
ranged from 15 to 45 years.
b)
Seventy infertile female with a medical history of primary or
secondary infertility due to hyperprolactinemia were studied.
c)
Thirty five patients were investigated for levels of estradiol .
2.1.2. Samples:
A single fasting blood sample was collected from each subject. From all
patients 5ml. of the venous blood samples were collected in the morning (8‫ـ‬
10a. m.), allowed to clot and immediately centrifuged for 10 minutes and
stored at - 20°C until analyzed.
20
2.1.3. Equipment:
i- Photometer: Biosystems BTS – 310 Photometer.
ii- Pipettes: Manual pipette was used for pipetting samples and standards
at the beginning of the assay, and automatic pipettes delivering 200µ,
500µ and 1ml were used for subsequent reagent additions.
iii- Vortex mixer: GENIE 2.
iv- Test tubes with round bottomed 12 ×75mm glass.
v- Magnetic racks and separators compatible with 12 ×75mm test-tubes.
vi- 37°C water bath.
vii-Refrigerator.
viii-
Timer.
ix- Various glassware including measuring cylinders, beakers and
reagent bottles of varring capacities.
2.1.4. Reagents:
2.1.4.1 Reagents for prolactin estimation:
Prolactin Enzyme Immunoassay (EIA) standards, prolactin EIA Enzyme
labeled antibody, prolactin EIA magnetic antibody EIA substrate reagent,
EIA substrate buffer, prolactin EIA assay buffer, prolactin EIA wash buffer,
EIA stop buffer, prolactin EIA QC sample and De-ionized or distilled water.
2.1.4.2. Reagent for Follicle Stimulating Hormone (FSH) estimation:
FSH EIA standards, FSH EIA Enzyme Lapelled Antibody, FSH EIA
magnetic antibody, EIA substrate reagent, EIA substrate assay buffer, FSH
EIA assay buffer, FSH EIA wash buffer, EIA stop buffer, FSH EIA QC
sample and de-ionized or distilled water.
21
2.1.4.3. Reagents for Luteinising Hormone (LH) estimation:
LH EIA standards, LH EIA enzyme Labelled Antibody LH EIA magnetic
labeled Antibody, EIA substrate reagent, EIA substrate Buffer, LH EIA
wash Buffer, EIA stop Buffer, LH EIA Internal QC sample and De-ionized
or Distilled water.
2.1.4.4. Reagents for Estradiol (E2) estimation:
E2 EIA standards, E2 EIA Antiserum, E2 EIA Enzyme Label, E2 EIA
separation Reagent, E2 EIA wash concentrate, EIA substrate Reagent, EIA
substrate Buffer, E2 EIA QC. sample, E2 EIA wash concentrate and Distilled
water.
2.2. Methods:
Estimation of prolactin (PRL), Follicle stimulation Hormone (FSH),
Leutinizing hormone (LH) and Estradiol (E2) were carried out by
appropriate Enzyme – linked immuno sorbent assays (sawlat sufi and
Maggie hayes Immunoneterics UK 2004).
2.2.1. Prolactin estimation:
The enzyme immunoassay (EIA) system was developed for the
measurement of prolactin (PRL) in human serum or plasma.
The time required to complete an assay is approximately 3 hours. The
concentration range covered by standards was 0-2400 ml IU/L (WHO IRP
84 1500) with a minimum detectable dose of approximately 20 ml IU/L by
immunometrics (UK) Ltd.
2.2.1.1. Prolactin EIA Assay Procedure:
(a) Step1 reaction: Immuno extraction of prolactin
i- 100µl standard or sample were pipetted into test tubes.
22
ii- 100 µl working suspension of prolactin EIA Magnetic Antibody were
added at room temperature by using a 100µl repeating multi-dose
pipette.
iii- The tubes were covered with aluminum foil and briefly vortex mixed.
Then the tubes were incubated in a water bath at 37°C for 15 minutes.
Wash step I
i- The assay tubes were removed from the water bath.
ii- 500 µl diluted prolactins EIA wash Buffer was added to tubes at room
temperature using repeating 500 µl multi-dose pipette, and briefly
vortex – mixed.
iii- The rack of tubes was placed onto a magnetic base for 5–10 minutes.
All the tubes were in contact with the surface of the magnetic base
until all magnetic particles had been sedimented.
iv- The supernatant liquid was decanted from all tubes by inverting the
separator over a sink (Keeping all tubes in contact with the magnetic
base). Immediately the inverted separator was placed on absorbent
paper and removed remaining droplets in tubes by gently tapping the
tubes in the separator on the paper.
v- The separator was returned to an upright position.
vi- The rack of tubes was removed from the magnetic base.
(b) Step II reaction: Labelled Antibody reaction:
i- After removing the rack of tubes from the magnetic base, 250 µl
diluted prolactin EIA Enzyme-labelled antibody was added at room
temperature.
ii- The tubes were covered and briefly vortex-mixed and incubated in a
waterbath at 37°C for 1 hour.
23
Wash step II (two washes):
i- The rack of tubes was removed from the water bath.
ii- 500 µl of diluted prolactin EIA wash buffer were added to the tubes
and briefly vortex-mixed at room temperature.
iii- The rack of tubes was placed onto a magnetic base for 5–10 minutes
until all magnetic particles had been sedimented.
iv- The supernatant liquid was decanted from all tubes and separator
returned to the upright position.
v- The rack of tubes was then removed from the magnetic base.
vi- Steps (ii) to (v) were repeated. It was essential to wash the magnetic
solid phase twice to remove all unbound labeled- antibody.
(c) Colour Development step:
i- 500 µl of substrate solution was added to all tubes at room
temperature.
ii- A substrate blank tube required for zeroing the colorimeter was
included in this addition.
iii- The tubes were covered with suitable film and briefly vortex- mixed.
iv- The tubes were then transferred to 37°C waterbath for 30 minutes.
v- The tubes were removed from the waterbath.
vi- 1 ml of EIA stop Buffer was added to the tubes.
vii-The tubes were stood in the magnetic separator for at least 10
minutes.
This sedimented all particles producing a clear solution for colour
measurement.
24
(d) Optical Density Measurement:
The optical density (OD) had been read at 550 nm within 24 hours of the
completion of the assay using a photometer.
2.2.2. Follicle stimulating Hormone estimation:
The enzyme immunoassay (EIA) system was developed for the
measurement of FSH in human serum or plasma. The concentration range
covered by the standards is 0–200 IU/L by Immunometric (UK) Ltd.
2.2.2.1. FSH EIA Assay Procedure:
All steps of FSH EIA procedure are similar to the steps of prolactin EIA
procedure (2.2.1.1).
2.2.3. Luteinising Hormone estimation:
The enzyme immunoassay (EIA) system was developed for the
measurement of luteinising hormone (LH) in human serum or plasma.
The concentration range covered by the standards is 0–150 IV/L (WHO IRP
80/552) by immunometric (UK) ltd.
2.2.3.1. LH EIA Assay Procedure:
All steps of LH EIA procedure are similar to the steps of prolactin EIA
procedure (2.2.1.1).
2.2.4. Estradiol estimation:
i- Principle:
The estradiol EIA direct assay is of a limited competitive type. Specific
agent is used to displace estradiol from binding proteins, thus making it
available for antibody binding.
25
The estradiol in samples, controls or standards reacts with flouorescein
labeled polyclonal antiestradiol antibody and then equilibrates with a fixed
amount of alkaline phosphate labeled estradiol.
An anti–fluorescein separate the estradiol/estradiol-antibody complex from
unbound components by magnetic sedimentation.
The magnetic particles are incubated with enzyme substrate solution for a
fixed time and the reaction ended by addition of a stop buffer. The amount
of colour produced is inversely proportional to the amount of estradiol
present in the sample.
The estradiol concentration of test samples is then interpolated from a
calibration curve.
ii- Estradiol EIA Assay Procedure:
Samples were analyzed by the procedure described below carried in
steps:
Step I (setting):
• 150µl of standard and 150µl of sample were pipetted into labeled
tubes using a manual 150µl pipette.
•
200 µl of Estradiol Antibody were added to each of the standard and
sample tubes using a 200 µl repeating multi-dose pipette at room
temperature.
• Tubes were covered and briefly vortex mixed.
• The tubes were then incubated in a waterbath at 37°C for 20 minutes.
Step II (Enzyme addition):
• 200 µl of enzyme-labeled Estradiol was added to the tubes using a
200µl repeating multi-dose pipette at room temperature.
26
• The assay tubes were covered with plastic, aluminum or other suitable
film. After mixing the tubes were transferred to the water bath and
incubated for 20 minutes.
Step III:
• 200 µl of separation reagent was added to the tubes (well shaken to
ensure magnetic particles were well suspended).
• The assay tubes were then covered and after mixing, the tubes were
transferred to the waterbath and incubated for 5 minutes.
Step IV:
• The assay tubes were removed from the waterbath.
• The rack of tubes was stood on the magnetic base for 5 minutes
ensuring contact with the surface of the magnetic separator to
sediment magnetic particles.
•
The supernatant was decanted out of the tubes by inverting the
separator over a sink (keeping all tubes in contact with the magnetic
base).
• The inverted separator was immediately placed on absorbent paper
and remaining droplets of supernatant in the tubes were removed by
gently tapping the tubes in the separator on the paper.
• Separator was then retuned to an upright position.
• The rack of tubes was removed from the magnetic base.
Step V (washing):
• 500 µl of diluted E2 EIA wash buffer was added to tubes and briefly
vortex-mixed.
• The rack of tubes was placed onto magnetic base for 5 minutes to
sediment magnetic particles at room temperature.
27
• The supernatant liquid was decanted out of the tubes by the same
procedure described in step IV and the separator was then returned to
the upright position.
• The rack of tubes was removed from the magnetic base.
All these steps for washing were repeated one more time. It was essential
that the magnetic solid phase was washed twice to remove all unbound
enzyme label.
Step VI (Colour development):
• 500 µl of substrate solution was added to the tubes. A substrate blank
tube was included.
•
The assay tubes were covered with a suitable film and briefly vortexmixed.
• The tubes were transferred to 37°C waterbath and incubated for 60
minutes.
• The tubes were removed from the waterbath.
• 1 ml of EIA stop Buffer was added to tubes and briefly vortex mixed.
• The rack of tubes was placed onto a magnetic base and stood on the
magnetic base for at least 10 minutes. This sediments all particles
producing a clear solution for colour measurement.
Step VII Optical density measurement :
The optical density (OD) of tubes had been read within 24 hours of
completion of the assay using suitable photometer at 550nm and 492 nm .
Step VIII : Calculation of results :
OD 550 nm values had been used to calculate most results .
The OD values can be processed using an appropriate automated calculation
program .
28
2.2.5 Survey :
A structured questionnaire was designed (Appendix 10). The aim and
purpose of the questionnaire was fully explained to participants. It was also
made clear that the information will be strictly confidential and only used for
the research purpose. Consent of each participant in the questionnaire was
obtained and the author thereafter filled and analyzed the questionnaire.
2.2.6. Study duration and area:
The investigations were carried out in the period extending from 12th March
to 18th August 2005 in the Reproductive Health Care Centre in Khartoum.
The selection of this centre was for two reasons:1- The centre was ready to collaborate in this research by allowing the
investigator to use their laboratory facilities.
2- It is one of the leading fertility centers in the country and visited by
many infertiles couples from different regions of Sudan. Part of this
study was also carried out in Asia Hospital in Omdurman.
2.2.7. Statistical analysis Procedure:Technique:
Data generated was subjected to statistical Package for Social Sciences
(SPSS). Means (± SD) were tested using t-test to calculate the difference
between two groups or more (Mead,B. and Gurany,R.W.(1983)).
29
CHAPTER THREE
Results
3.1. Measurement of serum hormones:
3.1.1 Serum prolactin levels:
Table (1) shows the serum level of the prolactin of infertile females. They
are grouped into three categories; moderately high, high and very high with
reference to the level of hyperprolactinemia. The values upto 550mIU/L
were related to normal,. The 1st group of 28 patients exhibited prolactin level
of 551-799 mIU/L, 2nd twenty patients with prolactin value from 800 – 1244
mIU/L, 3rd group – twenty- two ( n = 22) patients with prolactin value more
than 1245 mIU/L.
Serum prolactin mean levels of the three groups are highly significant
compared to control mean ± SE of 146.93 ± 21.6 and P= 0.001 in 1st and 2nd
groups. P=.000I in the 3rd group
Figure (4) illustrates the findings in table (1) serum prolactin level and the
control of the three groups, it is clear that the serum prolactin levels in the
three groups are higher than the control.
30
Table (1): Grouping of infertile female based on PRL levels
No. of
Groups
PRL level
t-test of PRL
P<
patients
(Mean ± SE) mIU/L
670.07±10.4*
G1
24.7144*
28
G2
21.9833**
20
959.0±27.5**
0.001
G3
15.7251**
22
1679. 7±95.1***
0.001
control
-
15
146.93±21.6
-
Keys:** = Highly significant
G1= Group 1 Moderately high PRL level .
G2= Group 2 high PRL level .
G3= Group 3 Very high PRL level .
31
0.01
1800
1600
1400
mIU/L
1200
1000
800
600
400
200
0
C
PRLC
PRLG1
G1
G2
PRLG2
G3
PRLG3
Groups
Fig. (4): Prolactin levels in infertile patients and
control
`KEY
C = Control Group
G1 = Group 1 = Moderately high PRL level .
G2 = Group 2 = high PRL level .
G3 = Group 3 = Very high PRL level.
32
3.1.2. Serum gonadotropin levels:
The serum levels of FSH and LH for the three groups of hyperprolactinemic
infertile females are shown in table (2) and illustrated in Figures (5) and (6)
respectively. The mean values of FSH and LH were compared with mean
values of FSH and LH in the control group. A consistent drop, in the levels
of both FSH and LH with increasing degree of hyperprolactinemia is
observed. The drop is not significant for infertile females in goup 1 with
moderately high prolactin level. However, it is significant (P< .01) for 2nd
and 3rd group whose hyperprolactinemia is almost equal to or over 1000
mlIU/L.
33
Table (2): Prolactin and gonadotropin in the three groups of
infertile females
Groups
959.0±27.5**
1679.7±95.1***
146.93±21.6
11.00±0.16*
8.00±0.14**
14.15±1.00
8.45±0.98*
6.5± 0.87**
10.33±0.45
28
20
22
15
(mIU/L)
Mean ± SE
(mIU/ml)
Mean ± SE
12.81± 0.87
(mIU/ml)
Mean ± SE
PRL
G3
FSH
G2
LH
G1
670.07±10.4*
8.92±1.09n.s
N
Control Mean
Key:
G3 = Group1 very high prolactin level
G2 = Group2 high prolactin level
G1 = Group3 moderately high prolactin level
N = Number of patients.
34
± SE
16
14
12
mIU/ml
10
8
6
4
2
0
C
G1
G2
Groups
Fig. (5): FSH levels in infertile patients and control
Key:
C = Control
G1 = FSH level in group 1
G2 = FSH level in group 2
G3 = FSH level in group 3
35
G3
12
10
mIU/ml
8
6
4
2
0
C
G1
G2
Groups
Fig. (6): LH levels in infertile patients and control
Key:
C = Control
G1 = LH level in group 1
G2 = LH level in group 2
G3 = LH level in group 3
36
G3
3.1.3 Comparing gonadotropin with PRL levels:
The
differences
in
gonadotropins in
relation
to
the
degree
of
hyperprolactinemia in the three groups are shown in figure (7). It is evident
that as the level of prolactin increases in these patients so drop the
15
gonadotropins in a consistent manner.
1800
1600
10
1400
1200
mIU/L
mIU/ml
1000
5
800
600
400
0
200
0
Control
G1
G2
G3
Groups
PRL
FSH
LH
Fig. ( 7): Control Prolactin, FSH and LH Levels in Infertile Patients
Key
G1 = Hormone level in group 1
G2 = Hormone level in group 2
G3 = Hormone level in group 3
37
3.1.4. Menstrual cycle pattern in hyperprolactinemia:
The 70 patients were grouped according to menses patterns into three
groups, regular cycle (RC), irregular cycle (IC) and amenorrhoea (AM). This
is shown in table (3) and illustrated in fig (8 ) A different pattern has
emerged when hyperprolactinemic are grouped according to menses
regularity. From the table it is clear that amenorrheaic patients were having
the highest levels of FSH, LH and prolactin, the patients with regular cycle
are
having
the
lowest
levels
of
gonadotropins
and
prolactin.
Oligomenorrhoeic (irregular cycles) levels of these hormones were found to
be medium between the two groups. The amenorrheaic patients represents
more than half of the patients (55.7%), while patients with regular and
irregular cycle patterns represented 21.4% and 22.9% respectively.
FSH is significantly lower in regular and irregular menses than it is in
controls whereas it is significantly higher in amenorrheaic patients than in
controls. This in fact stands in contradiction to pattern without consideration
of menstrual cycle patterns.
LH is also significantly higher in amenorrheaic patients than in controls.
However, this hormone level in patients with regular and irregular cycles is
closer to the control value.
38
Table (3) Levels of PRL, FSH and LH in patients with different
menstrual cycle patterns:
Hormones
RC
IC
AM
Control
932.21±2.1*
1038.6± 6.6*
1412.63±9.7**
146.93±21.6
10.59±1.4**
10.64±1.0**
18.36±2.8**
14.15±1.0
8.04±0.1*
10.58±1.9
16.60±2.0**
10.33±0.7
15
16
39
15
PRL
Mean ± SE
FSH
Mean ± SE
LH
Mean ± SE
No. of patients
P< .01
Key:
RC = Regular cycle
IC = Irregular cycle
AM = Amenorrhea
39
Regular
21.4%
Amenorrhea
55.7%
Irregular
22.9%
Fig. (8) Menstrual cycle patterns
40
3.1.5 LH/FSH ratio:Among the study group those who have had serum LH higher than serum
FSH were 15 with a percentage of 21.42% of the study population.
3.1.6. Serum estradiol levels:
It is clear that estradiol is significantly lower in infertile females compared
to the control group with a mean ± SE of 29.20 ± 3.0 Pg/ml, P< 0.0001.
3.1.7. Measurement of esradiol (E2), PRL , FSH , and LH in infertile
patients:
The relationship between estrogen status gonadotropins and prolactin levels
were investigated in some hyperprolactinemic patients. The mean values is
presented in table (4) and figure (.9,10 and11). Table (1) in appendix shows
detailed values.
A complex pattern emerges from the data and careful interpretation is very
much needed.
Table (4): Estradiol, prolactin and gonadotropins in hyperprolacinemic
infertile females:
N
Estradiol (pg/ml)
Mean ± SE
PRL (mIU/L)
Mean ± SE
Control
15
29.2± 3.0
146.93±21.6
Patients
30
2.2433 ± 0.6
1237.60 ± 99.1
41
LH (mIU/ml) FSH (mIU/ml)
Mean ± SE Mean ± SE
10.33±0.7
7.49 ± 1.8
14.15±1.0
11.47 ± 2.8
3000
9
8
2500
7
6
5
1500
4
1000
3
2
500
1
0
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Patients
PRL
E2
Fig. (9): Prolactin and estradoil
42
pg/mL
mIU/L
2000
6
9
8
5
7
mIU/ml
4
6
5
3
4
2
3
2
1
0
1
1
3
5
7
9
11
13
15
17
19
21
Patients
LH
Fig . ( 10 ):LH and estradoil
43
E2
23
25
27
29
0
pg/mL
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
pg/mL
mIU/ml
9
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
P a t ie n t s
FSH
E2
F ig . ( 1 1 ) : FS H a n d e s t r a d o il
3.2 Questionnaire data analysis:
Part of the study was carried out with a questionnaire constructed to obtain
insight
into
socioeconomic
the
incidence
status,
of
hyperprolactinemia
environmental
and/or
regarding
genetical
factors
age,
and
pathologies. The questionnaire was conducted in 70 patients who was
diagnosed to be infertile due to high levels of prolactin, representing about
18.5 of reported infertility in these clinics.
3.2.1 Age:
Of these 70 patients who have infertility due to high levels of prolactin, 23
were in the age range 15 – 25 years, representing 32.9%, 34 patients were in
26 – 35 yeas, representing 48.6% and 13 patients were in the age group 36 –
45 years, representing 18.5% (Figure 12). Tables of the questionnaire
included in the table 2 in appendix.
44
All the patients have been married over 5 years. The detailed duration of
marital life is shown in Figure (13).
45
36 -45
19%
15 - 25
33%
26 -35
48%
Fig.12: Percent age groups of the patients
46
11-15 ‫ﺳﻨﺔ‬
11%
Over 15
4%
About 5
52%
6-10 ‫ﺳﻨﺔ‬
33%
Fig13 Marriage duration
47
3.2.2 Environmental and genetical factors:
Patients in the study were found to come predominantly from central (Sinar,
WhiteNile and Gazira) and northern parts of the country beside Khartoum
(Figure 14).
A minor group of the patients are from the south, west and east. However
residency over the past 5 years is not consistent with the original regions of
patients (Figure 15). In this respect, the author intend to look into tribal
origins as well as residency if an interplay of genetical and environmental
factors are to be considered.
The questionnaire showed that about 25% of the patients have infertility
incidence among relatives (Figure 16).
48
North
23%
Central
30%
East
6%
West
10%
South
4%
Khartoum
27%
Fig. 14: Residential regions of the patients
49
North
17%
Central
30%
East
6%
West
7%
South
0%
Khartoum
40%
Fig.15: Area of residence during the last 5 years
50
Yes
25.71%
No
74.29%
Fig. 16: Family history of infertility
51
3.2.3. Pathologic factors:
Hyperprolactinemia was diagnosed after complaints of breast secretion
(galactorrhea) in about 34.3%. However, 66.7% of the patients were without
complaints. Examination of the medical history of the patients indicates that
about 23% of them received medical treatment for their hyperprolactinemia.
44% of the patients were actually on medications for other diseases when the
present investigation was done (fig 17, 18, 19, 20). Patients with
hypothyroidism accounted for 8.57% of the investigated group. 2.86% were
found to be hypertensive and receiving treatment for their hypertension. No
diabetic patients were among the study group.
Yes
44.29%
No
55.71%
Fig . 17 : Drugs used by hyperprolactinemic infertile females
52
Yes
8.57%
No
91.43%
Fig. (18) Thyroid diseases
53
Yes
2.86%
No
97.14%
Fig. (19): Hypertensive patients
54
Yes
0.00%
No
100.00%
Fig. (20): Diabetic patients
55
3.3 Incidence and prevalence of hyperprolactinemia in
Sudanese female patients infertility:
It was undertaken in this study to determine the incidence of
hyperprolactinemia in a group referred infertile Sudanese female in the
Reproductive Health Care Center (RHCC) in Khartoum. The investigation
covered the period from 2001 to 2005, and in Asia Hospital for years 2004 –
2005.( in table10,11 Appendix ). The choice of the duration was governed
by data availability in the RHCC and the life time in Asia Hospital ( new
centre).
A slight but a steady increase in the incidence of hyperprolactinemia is
observed. In 2001 hyperprolactinemic infertile females accounted for 14.32
of all infertile females visiting the RHCC. In 2005 they accounted for
18.35%. A similar increment is also seen Asia Hospital record.
56
CHAPTER FOUR
DISCUSSION
4.1 Female infertility:
Reproductive health is a state of complete physical, mental and social wellbeing in all aspects relating to the reproductive system and to its functions
and processes. Infertility is a world-wide problem affecting people of all
communities, though the cause and magnitude may vary
with geographical location and socio-economic status (Farley and Belesey
1988).
Infertility is a crippling problem to million of couples affected the world
over. It is estimated that globally between 60-80 million couples suffer
from infertility every year(World Health Organization 1996).
The relationship between changes in prolactin, reproductive hormones and
menstrual pattern in infertile females vary widely and there are geographic
variations in the deferent cases of infertility. The ovarian sensitivity to
prolactin is also extremely variable.
Many fertility centers in Sudan are visited by many infertile couples
including the Reproductive Health Care Centre in Khartoum, Asia Hospital
for IV.F. in Omdurman and Sudan Centre for Fertility.
4.1.1 Infertile patients with high levels of serum prolactin:
Differences in PRL, FSH, LH and estradiol levels of Sudanese infertile
females were revealed in this study when compared with controls. The
following observation are evident and deserve consideration and discussion:
1) Serum PRL is statistically significantly higher in infertile Sudanese
females (hyperprolactinemia).
57
2) Serum FSH and LH are lower than the control in the three groups of
infertile Sudanese females.
3) FSH/LH ratio in the control group is almost one, while it is much less
than one in 21.42% of the 70 patients of the infertile Sudanese
females.
In over two thirds of hyperprolactinemia patients did not suffer from
galactorrhea, this is in a good agreement with previous finding (Rosato and
Garofalo 2002) where galactorrhea appears in 30% of patients.
Infertile patients with hyperprolactinemia are arbitrarily grouped into three
categories according to the levels of serum prolactin. Significantly elevated
serum prolactin in these groups of infertile Sudanese females is indicative of
the role of prolactin in causation of infertility. This finding is in good
agreement with previous findings (Homburg 2003). The second point which
deserves consideration is that in the first and second groups mean levels of
follicle stimulating hormone and Lutenizing hormone were lower but not
significantly different compared to the control group. In the 3rd group the
mean levels of follicle stimulating hormone and lutenizing hormone is
significantly lower than the control groups. This also in good agreement
with previous findings (Mohammed 1992), where it was suggested that high
serum prolactin may inhabit FSH and LH secretion (Mohamed 1992;
ML'Hermite et al., 1978). It was also suggested that the elevated prolactin
not only suppress the pituitary gonadotropins secretion, but also disturb
follicular development and luteal function in the ovary (Lino1997).
Hyperprolactinemia
a.A
reduction
in
may
granulosa
cause
cell
anovulation
number
and
FSH
through:
binding
b. Inhibition of granulosa cell 17 estradiol production by interfering with
FSH action.
58
c. Inadequate luteinization and reduced progesterone
d. The suppressive effects of prolactin on GnRH pulsatile release.
Normal or low FSH or LH levels suggest a pituitary or hypothalamic
abnormality
(hypogonadotropic
hypogonadism).
Elevated
follicle-
stimulating hormone (FSH) or luteinizing hormone (LH) levels suggest an
ovarian abnormality (hypergonadotropic hypergonadism). (Master-Humter
and Heimen 2006). This may explain variation in gonadotropin levels in
hyperprolactinemia.
Hypothalamic
amenorrhea
is
associated
with
abnormalities in gonadotropin-releasing hormone (GnRH) secretion and
disruption of the hypothalamic-pituitary-ovarian axis. The condition often is
caused by excessive weight loss, exercise, or stress (Mitan 2004).
The presence of elevated serum lutenizing hormones (LH) with suppressed
follicle stimulating hormone (FSH) resulting in an increase LH/FSH ratio
has been observed in 21.42% of the 70 patients under study. This in fact is a
higher incidence than that reported in the literature which is 17% (Luciano et
al.,1984).
Increased LH/FSH ratio is suggestive of polycystic ovary syndrome (PCO)
which associated with hyperprolactinemia may be responsible for infertility.
This is also in good agreement with previous reports (Owecki and Sowinki
2004). where it was suggested that functional hyperprolactinemia in PCO
may be remarkably high.
4.2 Hyperprolactinemia and menstrual patterns:
Patients in this study were further categorized on bases of menstrual cycle
patterns as regular, irregular or completely missed menses i.e. amenorrhea.
There are unrevealed differences with regard to the levels of gonadotropins
in relation to hyperprolactinemia degree affecting ovulation. These
59
differences are manifested in different menses patterns varying from
irregular to amenorrhoea.
Amenorrhoeic patients with significantly elevated of PRL mean levels group
and elevated means of gonadotropins hormones may indicate ovarian failure.
In this case the amenorrhoea may be due to compounded factors including
ovarian failure on one hand and hyperprolactinemia on the other (MasterHunter and Heiman 2006).
Regularly menstruating female exhibit lower prolactin levels compared to
amenorrhoeaic patients. Hyperprolactinemia has been reported to result in
short luteal phase (Elsheiksh et al.,1984). Prolactin has been found to be
persistently elevated not only in many of the subjects with menstrual
abnormalities but also in some of those with apparently normal menstrual
cycles. This depends on the underlying cause of hyperprolactinemia as well
as the severity of the condition. The incidence of hyperprolactinemia was,
however, much higher among women with amenorrhea than among those
with normal cycle or with patients with oligomenorrhea showing an
intermediate frequency. Serum PRL in the present study was found to be
significantly higher in amenorrhoea as compared with normal menses, while
the level in oligomenorrhoea was found to be intermediate between
amenorrhoea and regular cycles, which is consistent with other authors
findings (Bahamondes et al.,1985).
However, it appears paradoxical to see a different patterns with
gonadotropins to the one reported earlier without considerations of menses
regularity. This paradox may be resolved if one considers the amenorrhoeaic
patients with the highest PRL levels. The gonadotropins are expected to
peak during mid-cycle during the ovulatory phase and return to lower than
baseline levels of follicular phase. When PRL is extremely high it affects the
–ve feedback loop operating on gonadotropins through an appreciably high
60
levels of oestrogen. But high PRL suppressed oestrogen production
consequently gonadotropin will remain high. This explanation is offered
because all estimations were carried on samples withdrawn during follicular
phase. This explanation is quite plausible in the light of the present results
where hyperprolactinemia in amenorrhoeaic patients has shown to exist with
significantly low oestrogen and elevated LH and FSH. An alternative
explanation could be premature ovarian failure.
Premature ovarian failure (POF) or is defined as consisting of the triad of
amenorrhea, hypergonadotropinism, and hypoestrogenism in women under
the age of 40 years. Circulating gonadotropin levels rise whenever ovarian
failure occurs because of decreased negative estrogen feedback to the
hypothalamic-pituitary unit. Gonadotropin levels sometimes increase even in
the presence of viable oocytes, but the explanation for such increases is
unclear. The peculiar finding in this context is the hyperprolactinemia.
It has been attempted to check the levels in ovulatry or luteal phases
particularly in regular cycles and irregular cycles to see if different patterns
emerge that are comparable to the ammendrrheaic situation but that was not
practical.
Hypergonadotropic hypergonadism (elevated FSH and LH levels) in patients
with primary amenorrhea is caused by gonadal dysgenesis or premature
ovarian failure. Turner's syndrome (45,XO karyotype) is the most common
form of female gonadal dysgenesis (Master-Hunter and Heiman 2006).
61
4.3 Serum estradiol levels:
In this study significantly lower levels of estradiol in patients with
hyperprolactinemia has been noticed. It suggests that hyperprolactinemia
may lead to hypostrogenism. This is supported by reports pointing to the fact
that prolactinoma is a risk for osteoporosis (Adler et al., 1998) a condition
well known as a consequence of lack of oestrogens. When patients with
hyperprolactinemia are not treated, a number of ramifications can result the
most significant of which is ostoporosis. Evidence-based analysis shows that
bone mineralization also can be affected by such problems as gonadal
dysgenesis and possibly adrenal dysfunction. The hypoestrogenism
associated with hyperprolactinemia is commonly assumed to be a potential
cause of osteopenia in premenopausal women, just as decreased estrogen is
associated with bone loss following menopause (Sanfilippo1999).
4.4. Questionnaire data analysis:
4.4.1. Age :
Over 80% of the patients are within the best period of reproductivity.
Regarding the age it is not surprising to find almost 50% of the infertile
patients with hyperprolactinemia to be between 25-35 years. This perhaps
is the age females will be very much concerned about their reproductive
health. Although hyperprolactinemia could very much be encountered in a
much younger age, complaints are not clinically presented due to a number
of factors. Among these is lack of symptoms such as galactorrhea and
amenorrhoea. Another reason is the social stigma of sending young
unmarried females to seek medical advice in obstetric and gynaecology
clinics. This in fact will result in unfavourable situation later in life because
hyperprolactinemia has adverse complications such as pre-menopausal
osteoporosis optical nerve disorders (Kaye 1996; Valdemarsson 2004).
62
4.4.2. Geographical and ethnic distribution:
The majority of the patients are from Central and Northern parts of the
country. Whether this is a true indicator or not can not be concluded from
such a sstudy. Only a cohort study of a bigger size and a longer duration can
give a faithful conclusion. However, this does not rule out the importance of
a pilot study like the present one.
That a minor group is from the south, west and east of Sudan may be
explained by the remotenss of these areas, the socioeconomic status
(travelling and lodging expenses etc..), health awareness and educational
level of the patients. Added to this the cultural taboos that encircle infertility.
In the developing countries, the incidence of induced-infertility is
relatively high; being higher among the less educated people
of low economic status. On the other hand, in developed countries or among
individuals of higher socio-economic status, the main causes of infertility are
genetical, anatomical, or endocrinological (Farley and Belsey 1988).
4.4.3. Environmental and genetical factors:
It is perhaps worth to follow the prevalence of hyperprolactinemia infertility
in certain regions of the country namely north and central Sudan with further
studies to investigate the underling causes and if these are genetical,
environmental or both. A genetical factor may be considered particularly if
consanguity marriages are not overlooked; which is a wide spread practice in
Sudan and this reflected in this study by a 25% incidence of infertility
among relatives of the patients invetigated. Underlying genetical causes are
not well established in female infertility as they are in male infertility.
Turner's syndrome (45,XO karyotype) is the most common form of female
gonadal dysgenesis(Master-Hunter and Heiman 2006). However, other
genetical causes may involve
63
Mutation of specific genes of enzymes, receptors, hormone response
elements and/or G-proteins.
Environmental estrogens (xenoestrogens) are a diverse group of chemicals
that mimic estrogenic actions. Bisphenol A (BPA), a monomer of plastics
used in many consumer products, has estrogenic activity in vitro. The
pituitary lactotroph is a well established estrogen-responsive cell.
BPA is found to mimic estradiol in inducing hyperprolactinemia in
genetically predisposed rats and that the in vivo action of estradiol and BPA
in F344 rats is mediated, at least in part, by increasing PRL regulating factor
activity in the posterior pituitary (Rosemary et al., 1997). BPA appears to
regulate transcription through an ERE, suggesting that it binds to estrogen
receptors in both the anterior and posterior pituitaries. The possibility that
BPA and other xenoestrogens have adverse effects on the neuroendocrine
axis in susceptible human subpopulations is not excluded (Rosemary et al.,
1997).
While it has long been known that female fertility is impaired by oestrogen
exposure, it is unclear whether environmental pollutants with weak
oestrogenic effects are sufficiently potent and prevalent to have biological
effects in humans (Joffe 2003).
Chemicals in tobacco smoke can alter important reproductive hormones
(such as estrogen, progesterone, and testosterone)(Florack et al., 1994).
4.4.4. Associated Patholigies:
Over 11% on medications are either receiving antihypertensive drugs or
being treated for hypothyroidism. Another important consideration is that
mild to moderately raised prolactin levels often present as one of the earliest
signs of an impending or existing hypothyroid state. In women
64
hypothyroidism is most often due to an autoimmune process (Hashimoto’s
Disease).
Perhaps more significant than the presence of a raised plasma prolactin
level, is the reason for this having occurred in the first place. Raised plasma
prolactin levels may occur as a consequence of a prolactin producing
pituitary tumor (adenoma) or other intracranial lesions. In addition, the use
of certain drugs: (tranquilizers, antihypertensives, antidepressants, thiazides
and narcotics) can elevate the blood prolactin level. Thus, it is possible that
in some of the studied patients the underlying cause of their
hyperprolactinemia is the use of certain drugs.
The sample size in this study is small and can barely give a thorough
information pertinent to which cause is predominant over others.
A cohort study is likely to validate this finding which will need time,
resources and collaboration of investigators from different disciplines.
4.4.5. The incidence of hyperprolactinemia in Sudanese females:
The prevalence and incidence of hyperprolactinemia bearing in mind all the
different causes contributing to its precipitation seems to be on the rise in
Sudanese females of child-bearing age. The % of hyperprolactinemic
patients seen in the RHCC between 2001- 2005 went up from 14.3% to
18.3%. whether this steady increase is a valid observation can not be
ascertained here; further investigations are needed taking into consideration
a bigger sample size and perhaps a longer duration ( if a particular persisting
factor is an underling cause).
65
Conclusions and recommendations:
It is concluded from the present investigation that hyprerprolactinemia
though not of high incidence ( less than 18%) is one of the primary causes of
infertility in Sudan. It is often accompanied by decreased gonadotropins.
In accordance with other studies, hyprerprolactinemia is found to be
responsible for menstrual cycle abnormalities with the dominance of
anovulation ( >50%). However, in some patients menstrual cycles are not
affected. It is worthwhile noting that this situation is only seen when
hyprerprolactinemia is not severe ( moderately high level < 800 µ IU/L).
Hypoestrogenism is also found in these patients confirming the idea that
untreated hyprerprolactinemia precipitate osteoporosis, presumably due to
the low estrogen levels rather than due to high prolactin levels.
An interplay of hormonal milieu seems to govern the outcome of the
condition in infertile females.
Recommendations:
•
More investigations are needed to resolve the complexity of the
issue.
• Attention should be given to the infertility problem in the country in a
coordinated manner.
66
• Socioeconomic, environmental and genetical factors as underlying
causes of infertility should be addressed and investigated through
multidisciplinary teams.
• Cohort studies should be encouraged and facilitated through
governmental bodies and the private sector, which will benefit future
work and planning regarding reproductive health.
67
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1.
Appendices:
Table (1) Measurement of esradiol (E2) and PRL , FSH , and LH in patients
PRL(mIU/L)
1208
2228
1175
1528
1368
1402
2800
1727
986
1254
2600
1077
1147
929
648
1425
1246
577
647
800
1317
1084
1104
1211
961
954
1678
625
958
763
FSH(mIU/ml)
0.8
3.2
6.8
7.00
1.8
3.2
2.2
5.8
5.4
0.9
1.2
3.5
1.6
1.7
1.6
1.6
4.9
7.7
2.6
1.00
4.4
0.5
0.3
5.4
2.00
6.4
1.1
5.1
0.7
4.00
80
LH(mIU)/ml
4.6
2.0
0.4
4.9
5.7
1.2
0.3
3.3
1.5
0.8
0.3
1.9
2.6
0.7
0.3
1.00
3.9
0.7
5.6
0.6
1.00
0.2
0.2
0.6
2.1
2.6
3.8
0.8
0.9
2.9
E2(pg/ml)
0.125
0.8
0.625
0.125
0.1
0.25
0.1
0.375
0.25
0.25
8.5
0.4
1.3
2.4
1.00
1.7
6.1
3.4
2.00
2.7
3.5
1.7
0.1
2.8
5.6
0.5
0.2
1.6
0.1
7.00
Table 2: Ages of patients (years)
Age groups
Frequency
%
15-25
23
32.86
26-35
34
48.57
36-45
13
18.57
70
100%
Total
Table 3: Marriage duration (years)
Years
Frequency
%
About 5
36
51.42
6-10
23
32.86
11-15
8
11.43
Over 15
3
4.29
70
100%
Total
Table (4): Presence and history of other types of disease in
hyperprolactinemic patients
Diseases
No. of patients
%
Hypertension
2
2.86%
Thyroid
6
8.57%
Diabetes
0
0
81
Table 5 : Menstrual cycle pattern
Frequency
%
Regular
15
21.4
Irregular
16
22.9
Amenorrhea
39
55.7
Total
70
100%
Table 6: Drugs used by hyperprolactinemia infertile female
Frequency
%
Yes
31
44.29
No
39
55.71
70
100%
Total
Table 7: Family history of infertility
Frequency
%
Yes
18
25.71
No
52
74.29
70
100%
Total
82
Table 8: Geographical distribution of patients
Origin
Frequency
%
Northern of Sudan
16
22.86
Eastern of Sudan
4
5.71
Western of Sudan
7
10.00
Southern of Sudan
3
4.29
Khartoum
19
27.14
Central of Sudan
21
30.00
70
100%
Total
83
Table 9: Area of residence during the last five years
Residence
Frequency
%
Northern of Sudan
12
17.15
Eastern of Sudan
4
5.71
Western of Sudan
5
7.14
Southern of Sudan
0.0
0.0
Khartoum
28
40.00
Central of Sudan
21
30.00
70
100%
Total
Table (10): The incidence of hyperprolactinemia in Sudanese female
patients infertility in reproductive health care center (2001 – 2005)
Hormone
Year
No. of patients
High level (abnormalities)
PRL
FSH
LH
PRL
FSH
LH
PRL
FSH
LH
PRL
FSH
LH
PRL
FSH
LH
2001
“
“
2002
“
“
2003
“
“
2004
“
“
2005
“
“
998
1051
1043
1202
1130
987
935
1384
1329
1472
1730
1699
975
941
925
143
241
313
174
245
240
141
303
258
239
412
482
179
236
303
84
%
14.32
22.93
30.00
14.44
21.68
24.31
15.08
21.89
26.93
16.236
23.82
28.36
18.35
25.07
32.75
Table (11): The incidence of hyperprolactinemia in Sudanese female
patients infertility in Asia Hospital. (2001 – 2005)
Hormone
Year
No.
of High
patients
level %
(abnormalities)
PRL
2004
29
5
17.24
FSH
“
32
8
25.00
LH
“
23
5
21.73
PRL
2005
172
32
18.60
FSH
“
165
43
26.06
LH
“
85
25
29.41
85