Practical Guide to Ovulation Induction Sonal Panchal, Chaitanya Nagori
Page numbers followed by b refer to box, f refer to figure, fc refer to flowchart, and t refer to table.
Acupuncture 130
Adenosine monophosphate 154
Adrenal androgen 107fc
Adrenal cytochrome, serine phosphorylation of 107
Adrenal function 40
Albumin, intravenous 172
Allergic reaction, local 162
Amenorrhea 19f
American College of Obstetricians and Gynecologists 41
American Society for Reproductive Medicine 106, 193
intraovarian 204
metabolism 107fc
Anorexia nervosa 20
Anovulation 19f, 20, 106
causes of 19
Antagonist 94t, 115
advantages of 94
agonist protocol 64
cycle 160
disadvantages of 94
protocol 64
advantages of 114b
Antibiotic therapy 176
Antibodies, development of 102
Anticoagulant 176, 177
therapy 176
Antihistamine 176
Anti-Müllerian hormone 7, 13, 27, 28, 32, 34, 64, 65, 90, 106, 129, 169
assays 30
clinical uses of 30
high 169
immunoassays 30
normal values of 30t
physiology of 13, 29
serum 12
values 13t
Antral follicle count 7, 25, 31, 32, 34, 60, 64, 72, 73, 73f, 75, 76
Anxiety 100
action of 50f
inhibitor 50, 137
Ascites 175, 176
Ascitic fluid
aspiration of 177
autotransfusion of 178
Aspirin 125, 130
low dose 46
Assisted reproductive technique 25, 53, 58, 63, 76, 89, 90, 112, 118b, 124, 135, 140, 157, 170, 187
Autoimmune disease 21
Basal body temperature 16, 183
Batch-to-batch inconsistencies 162
Beta thalassemia 100
Blood comp 175
Body mass index 21, 33, 34, 52, 60, 64, 72, 75, 76, 108
higher 143
low 169
Bologna criteria consensus 34, 124
Bone morphogenetic protein 169
Breast 43
cancer 92
Bromocriptine 43, 44, 47, 203
Buserelin 89
Cabergoline 204
Calcium, intravenous 173
Carbimazole 208
Central nervous system 204
dysmenorrhea 42
mucus 16
Cervix 42
Chemotherapy 21
Cholesterol, pathway of 182fc
Cirrhosis 100
Clomiphene 39, 46, 136, 185
citrate 39, 40, 4447, 51, 108, 110, 125, 128, 136, 161, 185
antiestrogenic effects of 42
challenge test 27, 31, 34
contraindications of 40
cycle 5, 10
indications of 40
resistant 51
risks of 43
failure 58
resistant 58
treatment, extended 43
Color Doppler 71, 74f, 84f
Computed tomography 203
Corpus luteal flow 84, 85t, 184f
high resistance 85
Corpus luteum 18, 18f, 83, 85, 91, 183
low resistance blood flow of 86f
Craniopharyngioma 20
Cryopreservation 61
Cyclic adenosine monophosphate 162
rupture 177, 178
subcapsular 117
Cystic fibrosis transmembrane conductance regulator 169
Danazol 176
Dandy-Walker malformation 43
Decapeptyl 89
Dehydroepiandrosterone 4, 107, 129, 154
sulfate 12, 44, 111
Deoxyribonucleic acid 129, 159
recombinant 60
Dexamethasone 47
Diarrhea 175
Disease Control and Prevention, centers for 26
Diuretics 176, 177
Donor oocytes 125
Dopamine 176, 177, 202
agonist 172
Dual stimulation 127
Dual trigger 145, 192
Dysmucorrhea 42
Dyspnea 175
Electrolyte 175
replacement 176
Embryo 42, 162
transfer 62, 78, 150
Enclomiphene 39
Endocrinal tests 27
Endocrine dynamic tests 31
biopsy 17, 41, 183
columnar epithelium 81
flow, secretory 185f
injury 84
receptivity 52
thickness 91
vascularity 82
vessels 17
volume 82
Endometrioma 124
Endometrio-myometrial junction 80f
Endometriosis 51, 54, 92, 124, 183, 194
Endometritis, chronic 84
Endometrium 42, 85f, 153
features of 79
ultrasound image 80f, 81f
Doppler features of 81
echogenic 85
optimum pretrigger parameters of 83t
secretory 86f
Epithelial growth factor 181
Estradiol 3, 8f, 28, 34, 91, 142
level 170
replacement 144
serum 10
supplementation 193
upregulates cystic fibrosis transmembrane conductance regulator 169
Estrogen 6, 17, 43, 44, 47, 103, 195
priming 125, 128
receptors 51, 181
Estrone-3-glucuronide 182
European Society of Human Reproduction and Embryology 34, 106, 124
Exogenous estrogen 42
Exophthalmos 208
low 13
optimal 13
Fetal outcome 53
Fibroid 92
Fluid aspiration 177
Follicle 1
B-mode ultrasound image of 77f
calculated volume of 79f
multiple 92
number per ovary 20, 34, 75, 169
optimum pretrigger parameters of 83t
stimulates 54
Follicle-stimulating hormone 2f, 3, 3f, 9, 2628, 34, 39, 50, 57, 58, 59f, 62, 89, 100, 109, 124, 125, 129, 150, 153, 154, 157f, 158, 161, 166, 182, 206
clinical benefits of recombinant 63
level, correlation of 27t
midcycle 139
ovarian reserve test, exogenous 27, 31
receptor genotype 169
recombinant 58, 60, 61, 61t, 62, 62t, 63, 65, 72, 112
secretion 2
serum 9
stimulates 58
Follicular maturity 10
Follistatin 7
alpha 63
beta 63
Frozen embryo transfer 54, 195
cycles 195
indications of 195
Galactorrhea 15, 44
Gastrointestinal tract 204
Glucocorticoids 43, 44, 108, 111, 125, 129, 172
Gonadal protection during chemotherapy 92
Gonadotropin 2, 4, 43, 45, 47, 54, 57, 58, 63, 108, 111, 135137, 185
bioactivity of 112t
cycles 126
dosage of 95, 125
low dose 137
therapy 58, 101, 102, 141
indications of 58
principles of 58
Gonadotropin-releasing hormone 2, 3f, 31, 39, 73, 94, 101, 125, 139, 157, 183, 202
agonist 43, 45, 89, 90, 94, 108, 114, 126127, 141143, 150, 172, 189
clinical application of 91
downregulation 196
flare protocol 125
mechanism of action of 89t
microdose flare 125
minidose luteal phase 127
protocols 90
stop protocol 125
trigger 144
analog 89
stimulation test 27, 31, 34
antagonist 43, 93, 94, 108, 113, 125, 127, 128, 130
protocol 170
deficiency 100
advantages of 101b
disadvantages of 102b
Goserelin 89
factors 7
hormone 103, 125, 129
actions of 129b
additional of 103
releasing factor 130
Hematoma 102
Hemorrhage, severe postpartum 20
Hirsutism 92
adrenocorticotropic 12,44
antidiuretic 168
release of 101
replacement therapy 21, 101
Human chorionic gonadotropin 43, 45, 54, 59f, 64, 72, 92, 101, 140, 141, 150, 162, 166, 182, 191
indications of 141
recombinant 67, 142
supplementation 184, 185
surge 141
characteristics of 141
Human menopausal gonadotropin 17, 29, 45, 57, 58, 61, 62, 95, 102, 112, 125, 137, 162, 170
exogenous 151
highly purified 62, 153
Hydrothorax 175
Hydroxyethyl starch 172
solution, intravenous 172
Hyperandrogenemia 107
Hyperandrogenism 20, 106
biochemical 20
clinical 20
Hyperinsulinemia 20, 107
inhibits sex hormone-binding globulin 107
Hyperplasia, congenital adrenal 106
Hyperprolactinemia 21, 101, 183, 202, 209
causes of 202
effect of 204
transient 204
Hyperstimulation 11
Hyperthyroidism 208, 209
Hypoestrinism, signs of 100
Hypogonadotropic hypogonadism 6, 19, 40, 58, 100, 103, 104, 140, 158
causes of 100
Hypophysectomy 20
dysfunction 144
dysfunction 20, 140, 183
failure 19
ovarian axis 2, 3f, 40, 52, 189
Hypothyroidism 203, 206, 209
isolated 101
subclinical 207, 209
In vitro fertilization 27, 54, 94, 104, 108, 125, 150, 168, 191
natural cycle 71, 125, 130, 136
In vitro maturation 54, 108, 118, 125
Indomethacin 177
Infertility 110b, 206
Infusion pump, use of 102
Inhibin 6
A 12
B 12, 28
Insemination, artificial 16
like growth factor 7, 50, 101, 129, 181
lowering drugs 108
insulin sensitizers 109
receptor, beta-chain of 107
resistance 14
sensitizer 29, 43
Intracytoplasmic sperm injection 72, 110
Intramuscular progesterone 187
Intrauterine insemination 40, 54, 58, 71, 114, 140, 168, 185, 191
Kallmann syndrome 20, 100
Killer cells, lymphokine-activated 182
Laparoscopic ovarian multi-needle intervention 116, 117
L-arginine 125, 128
Letrozole 50, 52, 54, 108, 109, 125, 128, 185
advantages of 51, 52
characteristics of 51
indications of 51, 51b
replace clomiphene citrate 54
Leukemia inhibitory factor 52, 181, 190
Leuprolide 89
disease 40
function tests 167
Low molecular weight 145
Luteal phase
defect 6, 40, 85, 85t, 144, 181, 184, 184f, 185f, 204
causes of 183, 183b
diagnosis of 183
less chances of 141
physiology 182
scan 71
Luteal serum progesterone levels 17
Luteal support, duration of 194
Luteinizing hormone 2, 2f4f, 5, 6, 29, 39, 50, 57, 65, 79, 85, 91, 94, 100, 106, 107, 125, 129, 136, 139, 140, 150, 154b, 157, 157f, 159163, 181, 208
action of 157f
addition of 95
ceiling 4, 65
detection kit 41
estimation 66
levels 158f
midcycle 16
physiology of 158
polymorphism 161
premature 89, 126
recombinant 141, 145, 172
releasing hormone 51
role of 157
serum 10, 27
stimulates 157
supplementation 65, 66, 126
suppression of 191b
surge 16
functions of 139
natural 139
therapeutic window of 158
window, concept of 5f
Mature follicle
features of 76
ultrasound image of 76f
color and pulse Doppler ultrasound image of 8f
Meningoencephalitis 100
Menopause 21
diagnosis of 9
premature 21
Menstrual pattern 27
Metformin 46, 47, 110, 110b, 111b, 171
after conception 110
in vitro fertilization 110
Methimazole 208
Miscarriage 43
Multilayered endometrium, blurring of outer margin of 85
Multiple follicular rupture 142
Muscular ventricular septal defect 43
Naperelin 89
Natural cycle in vitro fertilization, concept of 135
Nausea 175
New promising tests 184
Norethisterone 114
Obesity 124, 183
Oligoanovulation 204
Oligo-ovulation 20
Oliguria 175
Oocytes 153, 162
higher number of 61
Oral contraceptive pill 9, 46, 91, 111, 114, 126, 130, 195
Oral progesterones 187
Ovarian artery perfusion 83
Ovarian blood flow 31, 33
Ovarian cancer 43
Ovarian cyst, large 40
Ovarian drilling 6, 43, 47, 108, 115, 116b
advantages of 116
disadvantages of 116
laparoscopic 115, 116, 170
method of 117
Ovarian failure 21
causes of 21
premature 101
Ovarian hyperstimulation syndrome 29, 43, 54, 59, 65, 91, 101, 106, 135, 140, 161, 166, 185
classification of 166
clinical pathology of 168fc
management of 174t
medical management of 176b
moderate 167, 174, 175t
pathophysiology of 167
prediction of 168
prevention of 170, 195
primary prevention of 170
secondary prevention of 170b, 171
severe 34, 174, 175t
surgical management of 177b
treatment of 174, 176
types of 166
Ovarian hyperstimulation, controlled 53, 58, 136, 146, 161
Ovarian reserve 26
assessment of 9, 25
diminished 35, 129
test 26, 34, 34t
Ovarian response 13t
cost-effectivity of 65t
predictive index 33
calculation of 34t
predictors of 64, 65t
Ovarian size 176
Ovarian steroids genesis 9f
Ovarian stimulation, controlled 14, 57, 65, 150
controlled 190
mild 130
optimizing 173
Ovarian stromal blood flow 75
Ovarian stromal flow 74f
index 33
Ovarian suppression 11, 172
Ovarian surgery 124
Ovarian torsion 177, 178
Ovarian volume 31, 32, 60
Ovary 42, 154f, 167
three-dimensional ultrasound image of 73f
Ovulation 1, 7, 52
clinicopathology of 9
diagnosis of 15
induction 3, 95, 103, 104, 106, 107, 124, 135, 150, 157, 184, 185
drugs 27t
mechanism of induction of 50
physiology of 1, 58
rate 41, 41t
summarizing diagnosis of 18
trigger 139, 141
Ovulatory dysfunction 100, 209
Ovum 42
donation 21, 130
pick-up 32
Pain, abdominal 175
Paracentesis, abdominal 178
Peak systolic velocity 7, 33, 60, 72, 75, 76, 83
Pelvic infection 124
Perifollicular resistance index 77
Pituitary tumors 20
Plasma expanders 176
Pleurocentesis 178
Polycystic ovarian
morphology 13
syndrome 5, 10, 28, 29, 33, 39, 51, 59, 93, 101, 106, 107, 118, 118b, 144, 158, 161, 169, 185, 202, 203, 207
Polymorphism, receptor 124
Postchemotherapy 124
Postradiotherapy 124
Predictive index 34
Pregnancy 170
ectopic 177, 178
loss, recurrent 190, 193
multiple 43, 59
rate 61, 62
clinical 65
status of 176
termination 177, 178
Pregnanediol 3-alpha glucuronide 182
Pre-human chorionic gonadotropin follicle, Doppler features of good 77
Pre-in vitro fertilization androgens 125, 129
Premenstrual syndrome 42
Preovulatory period 154fc
Progesterone 7, 47, 91, 103, 142, 150, 153fc, 187, 189, 193, 195
effect of 153
gel 188
induced blocking factor 182
level 154fc
micronized 47
receptor isoform 194
replacement 144
role of 181b, 186
serum 11, 183
supplementation 184, 186
synthetic 187
vaginal effervescent tablets 188
Prolactin 202, 203
effect of 202
inhibiting factor 21
serum 15
Prolactinoma 203
Propylthiouracil 208
Prostaglandin synthetase inhibitor 176
Prostatic cancer, metabolic 92
Protein contamination 162
Psychiatric disorders 100
Puberty, precocious 92
Pulsatile gonadotropin-releasing hormone therapy 101
Pulsatility index 83
Pulse Doppler 17, 71, 74f, 84f
Pulse repetition frequency 78
Pyridostigmine 125, 130
Radiotherapy 20, 21
Randomized control trials 46, 109
Reactive oxygen species 193
Renal failure 100, 175, 203
Reproductive techniques 9
Resistance index 60, 75, 76, 83, 85
Respiratory distress syndrome, acute 167
Royal College of Obstetricians and Gynaecologists guidelines 41
Sarcoidosis 100
Secretory endometrium, ring sign of 19f
Segmental uterine artery perfusion 81
Serine phosphorylation 107, 107fc
Sex hormone-binding globulin 12, 130
Sheehan's syndrome 20
Single nucleotide polymorphism 28, 161
Skin reaction 102
Society of Assisted Reproductive Treatment 26
Spiral artery flow 84, 85t
Steroid production, pathway of 154f
Steroidogenic acute regulatory 182
Stimulation, mild 135, 137
Strenuous exercises 100
Stress 20, 100, 183
Stromal blood flows, inclusion of 74
Stromal flow index 169
Stromal resistance index 169
Substitutional therapy 58
Surrogate luteinizing hormone surge, indications of 140
Syphilis 100
Tachycardia 208
Testosterone, serum 12
Three-dimensional manual multiplanar view techniques 73
Three-dimensional power Doppler 71
ultrasound image 75f
Three-dimensional ultrasound 71
acquired virtual organ computer-aided analysis 79f
calculated ovarian volume 74f
Thromboembolism 177
Thrombophlebitis 102
disorders 206
drugs for 209
function tests 40
hormone 203
stimulating hormone 203, 206
mechanism of action of 207fc
serum 15, 203
Total ovarian vascularization index 33
Transvaginal hydrolaparoscopic drilling 116, 117
Transvaginal sonography guided aspiration 177
Transvaginal ultrasound 32
guided follicular aspiration 117
Triptorelin 89
Tuberculosis 100
infections like 21
Turner's syndrome 21
gonadotropin theory 5f, 65, 153, 153fc
hormone theory 158
Two-dimensional manual multiplanar view techniques 73
Ultrasound 17, 71, 184
guided transvaginal ovarian needle drilling 116, 117
Urinary follicle-stimulating hormone 58, 61, 61t, 62t, 65
Urine output 175
Uterine artery flow waveform, high resistance 83f
Vaginal progesterone 188
Vaginal, absorption of 189f
Vascular endothelial growth factor 13, 52, 115, 139, 142, 167, 181, 182
Vascular permeability factor 168
Vascularity flow index 75f
Vascularization index 75f
Venous thrombosis 175, 167
Virtual organ computer-aided analysis 74f, 78
Vomiting 175
Weight loss 108
Zuclomiphene 39
Chapter Notes

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Physiology of OvulationCHAPTER 1

Chaitanya Nagori,
Sonal Panchal
Ovulation is a very complex phenomenon and its clear understanding is essential to understand the abnormalities of the same. Ovulation will be discussed here under following heads:
  • Physiology of ovulation
  • Endocrinal control of ovulation
  • Tests to detect ovulation
  • Causes of anovulation.
Detailed understanding of physiology of ovulation is very essential to manage ovulation dysfunction, which is one of the important causes of fertility problems. Ovulation occurs from the ovaries. Each oocyte is surrounded by a number of cells to create a follicle. When the menstrual cycle begins one, or maybe even a few, primary oocytes begin to grow larger and the follicle cells increase in number and cause the follicle to grow larger too. Usually, some of the developing oocytes will degenerate and only leave one follicle that will mature, but every once in a while two, or even more, follicles will mature. As a follicle reaches maturity, the primary oocyte completes its first meiotic division and becomes a secondary oocyte. Very soon after that the follicle ruptures, and the secondary oocyte is released into the fallopian tube, even though the second meiotic division has not occurred yet. The release of a secondary oocyte from the ovaries is called ovulation.1
During intrauterine life of a woman, there are 7 million oogonia that reduce to only 1 million at the time of birth.2
This number is further reduced to only 300–400 thousands at the menarche. At the time of menopause, only 1,500 ova are available. During reproductive age only 400 ova reach up to ovulation, i.e. only 0.1% of the total available ova at menarche, reach up to ovulation and 99.9% become atretic.3
Ovulation occurs once in a month in majority of the normal women. It is the result of the integrated and synchronized succession of hormonal action and morphological changes in hypothalamus, pituitary and ovary. Autocrine and paracrine factors also take part in ovulation induction. So neuroendocrine 2complex system regulates ovulation and menstrual cycle.
Figure 1.1 explains the influence of different hormones on ovaries and endometrium. Grossly the gonadotropin-releasing hormones (GnRHs) released from the hypothalamus, control the gonadotropin [luteinizing hormone (LH) or follicle-stimulating hormone (FSH)] secretion from the pituitary and these gonadotropins finally control the estrogen and progesterone secretion from the ovaries. These steroids are responsible for the cyclical changes in ovaries and uterus. The entire control system is thus called hypothalamic-pituitary-ovarian axis. This hypothalamic-pituitary-ovarian axis should be intact and synchronized for normal ovulation (Fig. 1.2).
Gonadotropin-releasing Hormone
A decapeptide is synthesized and secreted from the neuronal endings in anterior and mediobasal part of the hypothalamus. It is released from hypothalamus and acts on the pituitary. GnRH traverses through hypothalamic-hypophyseal portal system. This is a compact blood vessel system and so it is not detected in circulation and it releases FSH and LH from the pituitary. Its secretion is pulsatile in nature and stimulates pituitary to secrete more LH than FSH. It is always correlated with LH pulse. Each pulse is of 60–90 minutes. It is chiefly influenced by ovarian steroids.
zoom view
Fig. 1.1: Hormonal and morphological variations during the menstrual cycle—diagrammatic representation.4 (LH: luteinizing hormone; FSH: follicle-stimulating hormone).
zoom view
Fig. 1.2: Diagrammatic representation of hypothalamic-pituitary-ovarian (HPO) axis.5 (GnRH: gonadotropin-releasing hormone; LH: luteinizing hormone; FSH: follicle-stimulating hormone).
Opiates, catecholamines and neuropeptides, all have an influence on GnRH secretion. There is enormous release of GnRH before the LH surge and then under the effect of progesterone, the frequency of the GnRH pulse decreases to only one in 8–12 hours.6 GnRH is suppressed by inhibitory factors till puberty.
Clinical Application for Ovulation Induction
  • It is used for ovulation induction in hypogonadotropic hypogonadism. But unfortunately it is not yet widely available.
  • GnRH agonist (GnRHa) can be used for downregulation in in vitro fertilization (IVF).
  • GnRHa as short protocol is used for endogenous FSH and LH surge for ovulation induction.
  • GnRHa is used for ovulation trigger to elicit endogenous LH surge.
  • GnRH antagonist is used for prevention of LH surge in intrauterine insemination (IUI) and IVF cycles.
Follicle-stimulating Hormone
Follicle-stimulating hormone is secreted from anterior pituitary. FSH secretion is controlled by the feedback system though estradiol (E2) and progesterone. When corpus luteum undergoes atresia or regression just before the menstruation, the FSH secretion increases markedly. It acts on the preantral and antral follicles and helps in recruitment and stimulation of the graafian follicle to make it dominant. It also enhances the conversion of androgens to estrogen though the pathway of aromatase enzyme. Secretion of FSH is governed by estrogen. It frees the oocyte from follicular attachment and converts plasminogen into plasmin. It helps in development of LH receptors for ovulation and for the luteal phase action. It has a definite minimum value, below which it is not sensed by the follicles. This is called FSH threshold. This value is different for every individual. Second peak of FSH is in the midcycle. It has been observed in animals that this second peak of FSH is must for successful ovulation. If this peak is absent, it may cause luteinized unruptured follicle (LUF). But its role in human is not clear. This is so because in humans when surrogate trigger is used in form of LH or human chorionic gonadotropin (hCG), it acts successfully even in absence of FSH. Though the second FSH surge may select the follicles for the next cycle.
Clinical Application
  • It is responsible for LH expression after the follicle size of 10–12 mm. This means FSH rescues the follicle to maturity. So there is no need to increase the dose of FSH for follicular growth after 10–12 mm size. Instead supplementation of LH will grow the follicle.74
  • Follicle-stimulating hormone secretion decreases during periovulatory period because of progesterone, but bioactivity is highest during this period.
  • Granulosa cells have androgen receptors. Androgen acts on the immature cells and potentiates action of FSH, e.g. aromatase inhibitor that increases androgen and dehydroepiandrosterone (DHEA) also potentiates the action of FSH.
  • Only 1% of the LH receptors are required for folliculogenesis and so in spite of downregulation with GnRHa in long protocol, the follicle continues to grow.
  • Follicle-stimulating hormone can stimulate paracrine signal that sustains thecal androgen synthesis. So FSH is capable of ovarian androgen synthesis.7
Luteinizing Hormone
Luteinizing hormone is another hormone secreted from anterior pituitary. In the first half of the cycle, its level is low and secretion is pulsatile. High levels of LH are detrimental to ova. This can be explained by the LH threshold and LH ceiling concept.
Luteinizing hormone ceiling and LH threshold:8 Figure 1.3 indicates that threshold level, i.e. certain minimum amount is required for growth of follicle. But if the level goes beyond a certain level, it hampers the growth of the follicle. This is known as ceiling effect and that level of LH is called the LH ceiling level. So LH level required for follicle maturation should be between the threshold and the ceiling level. This is known as LH window9 (Fig. 1.4).
Luteinizing hormone causes luteinization of granulosa cells. LH acts on theca cells to produce androgen. With the rising estrogen levels with developing follicle, when the FSH gets a negative feedback, LH gets a positive feedback. LH permits final maturation of follicle forming secondary oocyte and first polar body. It causes disruption of cumulus-oocyte complex and rupture of the follicle. LH surge should be between 14 hours and 27 hours after E2 peak for ovum maturation for rupture and LH surge lasts for 48–50 hours. The two-cell-two hormone theory explains the role of LH in ovarian steroidogenesis10,11 (Fig. 1.5).
Both the gonadotropins—FSH and LH as well as both the cells, i.e. granulosa cells and theca cells are required for maturation of ova. LH stimulates theca cells to produce androgen from cholesterol.
zoom view
Fig. 1.3: Luteinizing hormone (LH) ceiling and LH threshold—explanation.
zoom view
Fig. 1.4: The concept of luteinizing hormone (LH) window.
zoom view
Fig. 1.5: Diagrammatic explanation of two-cell-two gonadotropin theory.
Androgen from theca cells go to granulosa cells where under the influence of FSH, androgen is converted into estrogen by aromatase activity. So both gonadotropins and both the cells are required for folliculogenesis.
Clinical Application
  • Luteinizing hormone in clomiphene citrate cycle: Clomiphene citrate stimulates FSH and LH from the hypothalamus. This LH may reach the ceiling level especially in polycystic ovarian syndrome (PCOS) and is detrimental to ova. The level of LH is therefore checked on day 8–10 of the cycle and if it is more than 10–12 IU/mL, it is less likely that clomiphene can give pregnancy as the follicle quality deteriorates due to high LH.12
  • Luteinizing hormone in PCOS: There is tonically high level of LH that causes abnormal oocyte maturation. There is also premature luteinization that decreases implantation because of progesterone 6secretion. So chronic low-dose protocol and antagonist protocol in PCOS gives very good pregnancy rate. High level of LH causing ceiling effect is clearly seen in PCOS.
  • Luteinizing hormone in downregulated cycle: In a downregulated cycle, there is low level of LH. When it reaches below the threshold level, the growth of the follicle is hampered. Low level of LH causes poor luteal conversion and so luteal phase defect (LPD) is very common. To compensate this loss, hCG works very well as luteal support but ovarian hyperstimulation syndrome (OHSS) should be kept in mind in PCOS patients.
  • Luteinizing hormone and ovarian drilling: By drilling, we are destroying stroma, i.e. theca cells. So androgen production is decreased and this will have negative feedback to hypothalamus and pituitary to decrease LH secretion. This decrease in LH will revert the LH/FSH ratio and spontaneous ovulation is established.
  • Luteinizing hormone in hypogonadotropic hypogonadism: LH is must for follicular development as it is discussed in two-cell-two gonadotropin theory. Without LH follicular growth is present but estrogen level is low.
  • Luteinizing hormone in poor responder: In poor responder rather than increasing dose of FSH, LH or hCG can be added for rescue of the follicle after 10 mm size. Here LH receptors have developed and it can rescue the follicular growth. But dose of more than 150 IU can have a LH ceiling effect.
It is secreted by granulosa cells of the follicles under the influence of FSH as explained in the two-cell-two hormone theory. It is also produced from androgen by aromatization as discussed earlier. This means for the production of estrogen, androgen is essential and androgen is produced in theca cells under the effect of LH.
High level of estrogen causes negative feedback mechanism and decreases FSH and LH. But when estrogen reaches to certain peak, it causes positive feedback mechanism and causes rise of FSH and LH. This rise causes LH peak and second FSH peak (surge). But LH peak is more intense than FSH peak.
Estrogen prepares the endometrium and is essential for development of progesterone receptors. Estrogen concentration is at their lowest at menstruation and rises to maximum at the preovulatory phase. Following ovulation, the estrogen level falls and then again rises in the luteal phase with the activity of corpus luteum.
Clinical Application
  • Because of negative feedback mechanism, FSH level decreases after rise of estrogen from developing follicle. This decrease in FSH causes monofollicular development of the follicle. Letrozole does not abolish this negative feedback unlike clomiphene citrate and so letrozole gives monofollicular development.
  • Exogenous estrogen is given for preparation of endometrium in premature ovarian failure (POF) patients.
Other Factors
Inhibin (Fig. 1.6)
Inhibin is also produced by granulosa cells. It has two dimers, inhibin A and inhibin B and both differ in their secretion pattern. Inhibin B rises in follicular phase and is inhibitory to FSH (similar to estrogen). Inhibin A rises in periovulatory period and luteal phase.7
zoom view
Fig. 1.6: Fine tuning system of HPO axis (short loop and long loop feedback systems).
Clinical application: Inhibin B is assessed for measuring ovarian reserve but anti-Müllerian hormone (AMH) and antral follicle count (AFC) are more reliable.
Activin and Follistatin (Fig. 1.6)
These increase secretion and promotes action of FSH, and lead to follicular growth and inhibit androgen production. Follistatin being an activin-binding protein, and neutralizes activin bioactivity.
Growth Factors
Insulin-like growth factor (IGF), IGF-2, IGF-binding protein (BP) 1–6, transforming growth factor (TGF), TGF-α, TGF-β, epithelial growth factor (EGF), etc. are the growth factors for the follicle growth. Activity of IGFs can be counteracted by IGF-BPs. Receptors to these are present in ovaries and promote androgen production. All these factors passively help the growth of the follicle (Fig. 1.6).
Ovulation occurs after 34–36 hours of onset of LH surge. Onset of LH surge correlates with perifollicular blood flows. Pre-LH surge perifollicular resistance index (RI) between 0.4 and 0.48 and peak systolic velocity (PSV) of 10 cm/sec are the indicators of a mature follicle on Doppler ultrasound13 (Fig. 1.7). Ovulation occurs 10–12 hours after LH peak (Fig. 1.8). Estrogen peak initiates LH peak and ovulation occurs after 24–26 hours of estrogen peak. Inhibitory activities are high before and after ovulation. But it is important to remember that apart from these hormones, normal levels of prolactin, thyroid and androgen are also essential for normal ovulation.
Human chorionic gonadotropin plays a major role in inducing influx of blood within follicles. Follicular PSV rises markedly with the rise in the LH levels at the time of preovulatory LH surge.15 Because of this physiology, when PSV of the follicle is high, it means that the follicle is likely to rupture early, IUI should be done earlier than the conventional protocol of 34–38 hours.
Luteinizing hormone induces the secretion of progesterone from luteinized granulosa cells (Fig. 1.9). Progesterone rises before ovulation and this rise is because of rise of FSH. It has a triggering role for high GnRH which may give LH surge.8
zoom view
Fig. 1.7: Color and pulse Doppler ultrasound image of a mature follicle.
zoom view
Fig. 1.8: Estradiol (E2) and LH fluctuations around ovulation.14
It helps in increasing proteolytic enzymes for rupture. Progesterone production from corpus luteum keeps on rising till the midluteal period (7–8 days postovulation), when its level is at its peak. Rising progesterone has a negative feedback on the pituitary gonadotropins and decreases the frequency of LH pulse. During this phase, FSH is synthesized but not secreted. It is stored for release when corpus luteum fails and estrogen and progesterone levels decrease markedly. Decrease in LH causes demise of corpus luteum and decrease in progesterone secretion towards the end of the cycle, just before menstruation.
Progesterone prepares endometrium for implantation by expressing genes needed for implantation. hCG of pregnancy rescues corpus luteum and progesterone secretion is maintained till the trophoblastic and placental hormones take over.
Clinical Application
  • Preparation of endometrium occurs with progesterone along with estrogen in POF patients.
  • Human chorionic gonadotropin can be used to rescue corpus luteum as a luteal phase support.
  • Luteal support with progesterone is given to the patients at high risk of LPD, especially those on agonist cycles and antagonist cycles.9
zoom view
Fig. 1.9: Ovarian steroids genesis.6
For the diagnosis of ovulatory dysfunction, it is extremely important to advice for precise investigations depending on the history of the patient. It helps the clinician to reach to the correct diagnosis. Unnecessary investigations may give no relevant information but may create more confusions.
Profiles and packages are useless in infertility and are expensive in clinical practice. In infertility management, specific investigations are required at specific times for correct interpretation. So one has to be very precise when requesting for investigations. The routinely required investigations for infertility and assisted reproductive techniques (ART) are discussed here.
Serum Follicle-stimulating Hormone
  • Day 2–3 serum FSH and serum LH for diagnosis of PCOS: High LH indicates anovulation and it is very evident from the history. Therefore, ratio of LH/FSH is not required for diagnosis of PCOS. Both high LH and PCOS can be diagnosed by an ultrasound scan done on day 2–3 of the menstrual cycle.
  • Serum FSH for diagnosis of menopause: FSH of greater than 40 IU/mL indicates ovarian failure. Oral contraceptive (OC) pills can be used to induce withdrawal bleed in these patients to confirm menopause. When the patient does not have a withdrawal bleed after giving progesterone for 3–5 days, it indicates she is menopausal. Ultrasound shows hardly any follicles and ovaries are small.
  • Follicle-stimulating hormone for assessment of ovarian reserve: Day 2–3 FSH is done to know the ovarian reserve and also to predict the possibility of pregnancy. Higher the FSH, lesser is the chance for development of good quality follicles/ova, thus resulting in a lesser chance to achieve conception. FSH of less than 8 IU/mL is considered normal. Between 8 IU/mL and 12 IU/mL FSH, the values are 10considered high. But when these values are greater than 12 IU/mL, there is very little chance of retrieving good quality ova and therefore decreased chance of achieving pregnancy. Chances of pregnancy beyond FSH level of 15 IU/mL is rare.16 Though currently age, AFC and AMH are used for assessment of ovarian reserve and thought to be much more accurate and reliable for the purpose, than FSH. Utility of FSH as a marker for ovarian reserve has significantly reduced. This is because, AFC indicates the number of follicle available in that cycle for recruitment, whereas serum FSH level gives overall impression about the availability of the ova.
If the patient with low ovarian reserve is given DHEA for 3 months, the AFC may rise, but the FSH still remains high. In these cases, it would be worthwhile to initiate ovulation induction. AFC thus may be a better marker for patient counseling too. Moreover, repeated FSH assessment is of no significance, because it is the highest level of FSH that acts as the decision maker for the possibility of pregnancy. AFC is so far considered the most feasible and reliable parameter for assessment of ovarian reserve.17,18
  • Low FSH: Less than 5 IU indicates pituitary or hypothalamic cause for anovulation.
Serum Luteinizing Hormone
  • Serum LH for PCOS: Day 2/3 serum level of LH is not required for the diagnosis of PCOS. In PCOS, LH is high and this can be clinically judged by anovulation or oligo-ovulation resulting in delayed menstrual cycles. High LH, leads to high androgen can be diagnosed by high stromal volume.19,20
  • Serum LH on day 8–10 in clomiphene citrate cycle: Clomiphene citrate stimulates hypothalamus to secrete both FSH and LH as it blocks estrogen receptors. This high LH is detrimental to ova and gives poor pregnancy rate in spite of good ovulation rate. Therefore, when LH is more than 10–12 IU/mL on day 8–10 of a clomiphene cycle, the subsequent cycles should not be continued with clomiphene citrate, instead the ovulation induction should then be done with gonadotropins in these patients. This is more common scenario in PCOS patients.
  • Serum LH in downregulated cycle: In downregulated patients on long protocol, the LH on day 2–3 of the treatment cycle should be between 0.5 IU and 2 IU to confirm the adequacy of downregulation. Actually 0.1 IU/mL LH is sufficient for the development of follicle and so assessment of LH levels at this phase of cycle is questionable.
  • Serum LH less than 5 IU/L indicates pituitary or hypothalamic cause for anovulation, especially with low FSH.
Serum Estradiol
  • Follicular maturity: When the follicle is mature, serum E2 will be 150 pg/mL/follicle. If E2 is normal and endometrium is poor, it is a local cause for poor endometrium. But if the E2 level is low, the endometrium is usually poor, and may grow to normal when E2 level rises. Using color Doppler for the assessment of follicular quality can be a useful tool for the same because the flow parameters of the follicle can be correlated with the functional maturity of the follicle which indirectly indicates E2 levels. So E2 can differentiate between the causes of poor endometrium.
  • For poor responder: In IVF cycles, E2 is assessed on day 2 of the cycle to confirm downregulation. If it is greater than 80 pg/mL, it indicates a possibility of poor 11response to stimulation. This high E2 is because of high FSH that is present in poor responders and causes early recruitment of the follicle.
  • To assess ovarian suppression: In long agonist protocol IVF cycles, GnRHa is started from day 21 of the previous cycle. This downregulates pituitary and FSH and LH levels become very low. This also leads to low E2 levels (30–50 pg/mL). The stimulation is started only thereafter. If E2 level is more than 50 pg/mL, it is advisable to continue downregulation for 1 day or 2 days more before starting the stimulation. In patients on OC pills, E2 should be checked after 12–14 days after starting OC pills.
  • For suspected risk of hyperstimulation: In IUI cycles or in non-IVF gonadotropin cycles, hyperstimulation is suspected if E2 is more than 1,600 pg/mL on the day of trigger. This is common in PCOS patients. In IVF cycles, if E2 is more than 4,000 pg/mL on the day of trigger, hyperstimulation is suspected. Now with chronic low-dose protocol in IUI cycles and ultrasound-based scoring systems for deciding the stimulation protocols,21,22 extremely low OHSS rates are claimed. Though in IVF cycles with antagonist protocol and agonist trigger, OHSS can be almost completely prevented. Therefore, the strategy in PCOS patients should be antagonist protocol with agonist trigger.
Ovarian hyperstimulation syndrome can also be predicted by calculating the ovarian volume. If total ovarian volume of both the ovaries together is more than approximately 180 cc, OHSS should be suspected,23 whereas if it is less than approximately 110 cc the risk of OHSS is extremely low. We depend more on ovarian volume than on the E2 levels and withhold hCG trigger when combined ovarian volume of two ovaries is more than 180 cc.
Serum Progesterone
Progesterone is chiefly secreted by corpus luteum in 2nd half of the cycle. Otherwise it is an intermediate hormone for other steroid hormones during their production after conversion from cholesterol.
Normal values:
  • Follicular phase
: 1 ng/mL
  • LH surge
: 1–2 ng/mL
  • Ovulation
: 3 ng/mL
Midluteal serum progesterone, i.e. a week before menstruation is approximately 10 ng/mL. A level of greater than 3 ng/mL indicates ovulation. Therefore, if only ovulation is to be documented, repeated ultrasound scans are not required. In the same way, endometrial biopsy is not required to confirm secretory changes in endometrium and serum progesterone estimation suffices for the same.
Ultrasound with color Doppler is a better modality to assess the secretory phase of the cycle. It can diagnose luteal phase problems viz. LPD/LUF. So it is an inevitable conclusion that random serum progesterone has little value beyond documenting ovulation.
This investigation is done in all patients with hirsutism. It is increased due to 21-hydroxylase deficiency. Because of 21-hydroxylase deficiency in patients of heterogeneous carrier, late onset of congenital adrenal hyperplasia is observed. 17-hydroxyprogesterone (17-OHP) is weak androgen and manifests as hirsutism, acne, clitoral enlargement and menstrual irregularity at puberty.
Normal level of 17-OHP is less than 200 ng/dL. Whenever the level is between 200 ng/dL and 800 ng/dL, one should get the 12adrenocorticotropic hormone (ACTH) levels assessed in serum. Level of greater than 800 ng/dL is diagnostic of 21-hydroxylase deficiency. These patients are treated with dexamethasone 0.5 mg daily at bedtime.
Serum Testosterone
Normal value of serum testosterone is 20–80 ng/dL. Its normal production is 0.2–0.3 mg/day. Out of this 80% binds to sex hormone-binding globulin (SHBG) and 19% to albumin. Only 1% is free testosterone. Testosterone may be high in patients with PCOS and tumors. Tumor is suspected if there is sudden onset of symptoms due to increased testosterone. PCOS patients show gradual rise in testosterone levels. Routine estimation of serum testosterone is not required, as it does not change the line of treatment.
Androgens and their sources have been shown in Table 1.1.
Serum Dehydroepiandrosterone Sulfate
Dehydroepiandrosterone sulfate (DHEA-S) is exclusively secreted from adrenal glands. The normal value is 350 ng/dL. It increases with hyperprolactinemia. In patients with PCOS, prolactin and DHEA-S are a little high and do not require any specific treatment except the treatment of PCOS. This rise is secondary to estrogen because of anovulation.
TABLE 1.1   Androgens and their sources.
50% Peripheral conversion
25% Ovary
25% Adrenal
50% Ovary
50% Adrenal
90% Adrenal
10% Ovary
100% Adrenal
(DHEA: dehydroepiandrosterone; DHA: dehydroandrosterone)
Whenever DHEA-S is very high, it is due to adrenal tumor. DHEA-S greater than 700 µg/dL is accepted as a marker for adrenal dysfunction. This is rarely found and does not change the management. And ultrasound is a better guide to diagnose adrenal tumor. Serum testosterone and ultrasound suffices to rule out adrenal tumors. If 17-OHP is normal, there is no need to search for adrenal enzyme defect. Therefore, routine testing of DHEA-S is not required.
Inhibin A and B
Inhibin B is predominantly secreted by antral follicles. Low day 3 inhibin B level less than 45 pg/mL indicates poor response to superovulation and patient is less likely to conceive. Same thing can be judged by serum FSH, serum AMH or AFC and so routine use of inhibin B estimation is not justified. Inhibin A is secreted from preovulatory follicles. Both inhibin A and B are secreted from granulosa cells and regulate FSH by negative feedback mechanism. Routine estimation of inhibin A does not add any information to change the management of the patient.
Serum Anti-Müllerian Hormone
It is a member of TGF. It is produced from granulosa cells of small antral follicles. The expression of AMH is localized in granulosa cells of primary preantral and small antral follicles and has important role in human folliculogenesis. AMH expression in follicle decreases in antral follicle greater than 8 mm in size. It is now well established that serum AMH concentrations reflect the number of preantral and small antral follicles in the ovary, this would account for raised AMH levels found in 13both polycystic ovarian morphology (PCOM) and PCOS.24 In follicles undergoing atresia and in corpus luteum also, the AMH expression is completely lost.11 AMH levels in women are low until the age of 8, rise rapidly until puberty and decline steadily from the age of 25 until menopause, when AMH production ceases.
Physiology of Anti-Müllerian Hormone
Anti-Müllerian hormone has an inhibiting role in the ovary, contributing to follicular arrest.19 It lowers the sensitivity of follicles to FSH, which is required for normal folliculogenesis. In vitro studies have shown that the action of FSH in promoting follicular growth is counteracted by AMH.25 It has a steady level throughout the cycle except for a slight dip just after LH peak.26
Though some investigators have recorded cyclical fluctuations in AMH with rapid decrease in early luteal phase.27 This can be correlated with the follicular atresia before recruitment in the late luteal phase.
It can predict ovarian response. It can predict menopause by low levels. High AMH is very high in PCOS, and it predicts OHSS.
Anti-Müllerian hormone values and its interpretation for ovarian response have been shown in Table 1.2.
Anti-Müllerian Hormone and PCOS
The reported property of AMH to counteract the actions of FSH imply that the high production of AMH by polycystic ovary may have an important role in the pathophysiology of the syndrome.28 In anovulatory PCOS, the failure of follicle development is due to an intrinsic inhibition of FSH action and that this inhibition is due to an intrinsic inhibition of FSH action and due to the high concentration of AMH.29
TABLE 1.2   Anti-Müllerian hormone values and ovarian response.
Ovarian fertility potential
Optimal fertility
Satisfactory fertility
Low fertility
Very low/undetectable
High level
Increased ovarian stromal blood flow in PCOS may be because of over expression of vascular endothelial growth factor (VEGF) that modulates the permeability of theca cells and increased IGF-130,31 which in turn enhances gonadotropin-stimulated steroid production in granulosa cells and theca cells resulting in increased ovarian androgen production and subsequently increased AMH production.32
Anti-Müllerian hormone levels are two to three times higher in PCOS, than in healthy controls.33 This has been attributed not only to increased number of antral follicles but also to higher production of AMH per follicle in patients with PCOS as compared to size-matched counterparts from normal ovary.34 In addition, a positive correlation between AMH and both LH and testosterone serum concentrations in PCOS has been reported.35
Each individual follicle in women with PCOS produces significantly more AMH than its size-matched counterpart from a patient with normal ovary.34 Moreover, it has also been shown that metformin administration in PCOS patients is associated with reduction in AMH concentration in follicle and serum, suggesting that the measurement of AMH can be used to evaluate the treatment efficacy with insulin sensitizers.36
Anti-Müllerian Hormone and IVF
In IVF patients, serum AMH value can predict the response of ovary and so it is useful for counseling the patient. When calculated optimal AMH cutoff of less than 1.26 ng/mL 14was used to predict responses to controlled ovarian stimulation (COS), it was found to have a 97% sensitivity for predicting poor responses (<4 oocytes retrieved) and 98% accuracy in predicting a normal COS response.37
Nelson et al. suggested the AMH-based strategy for deciding the COS protocol.38
But we have found AFC as equally effective and more precise for prediction of ovarian response. With extremely low-serum AMH levels, moderate, but reasonable pregnancy and live birth rates are still possible. Extremely low AMH levels do not seem to represent an appropriate marker for withholding fertility treatment. Younger women are likely to have better pregnancy rates than their older counterparts with equally low AMH.39 Constant AMH and inhibin B levels suggest that neither AMH nor inhibin B is an accurate marker of ovarian response after low-dose gonadotropins ovulation induction in patients with PCOS.40 Mashiach et al. have also shown a relationship between follicular fluid AMH concentrations and the quality of embryos in patients with PCOS.41
All these references indicate that routine use of AMH does not help in managing non-ART patients. Only 1–2 mature follicles are required for superovulation with IUI and AMH will not help us in change the line of treatment. Poor responding ovary or low reserve ovary can be very precisely diagnosed by ultrasound.
Serum AMH estimation is indicated in:42
  • Predicting both over and under response in COS
  • Determining the most appropriate treatment regimen
  • Pretreatment counseling for couples to make an appropriate and informed choice
  • Predicting long-term fertility
  • Predicting the age of menopause
  • Predicting ovarian aging prior to or following chemotherapy/surgery
  • Screening for polycystic ovaries.
Clinical Applications of AMH, Apart from Fertility Assessment
  • To confirm presence of testicular tissue in children with low testosterone levels.
  • Differential diagnosis of intersex disorders.
  • In patients with bilateral nonpalpable gonads.
  • Females with granulosa cell tumors.
  • The inhibitory effect of AMH on folliculogenesis may in future be used for hormonal contraception.
Insulin Resistance
Now it has been proved that in patients of PCOS there is high androgen because of increased insulin level and insulin resistance. It indicates the severity of PCOS. Clinically waist circumference of 35 inches or 90 cm is predictive of abnormal endocrinology and metabolic function.
There are various methods to diagnose insulin resistance. But 2-hour glucose and insulin response is the one of the most reliable ones.
Two-hour glucose tolerance test (75 gm of glucose):
  • Normal
: <140 mg/dL
  • Impaired
: 140–190 mg/dL
  • Noninsulin-dependent diabetes mellitus
: >200 mg/dL
Two-hour insulin response:
  • Insulin resistance very likely
: 100–150 µU/mL
  • Insulin resistance
: 151–300 µU/mL
  • Severe insulin resistance
: >300 µU/mL
In clinical practice, patients with high insulin resistance are the ones who have 15tonically high LH levels require ovarian drilling. Alternatively they may also be benefitted by GnRH antagonist. Antagonist decreases LH levels, prevents premature luteinization and improves oocyte quality.
Patients who do not want immediate pregnancy are the patients in whom insulin sensitizer alone is the recommended line of treatment. It has been proved beyond doubts that insulin sensitizers do not have any direct effect on ovulation induction, though when given for 3–6 months, the improved insulin sensitivity, improved the hormonal milieu of PCOS patient, thus improving the chance of spontaneous ovulation and conception.
Three-dimensional (3D) ultrasound is a novel way to diagnose insulin resistance, based on assessment of stromal volume.43 But when volume ultrasound is unavailable, 2-hour glucose and 2-hour insulin test is the best alternative.
So for chronic anovulation only three investigations are required:
  1. Serum prolactin
  2. Serum TSH
  3. Insulin resistance.
Serum Prolactin
Prolactin is the hormone secreted by anterior pituitary and is regulated by prolactin inhibitory factor that is secreted from hypothalamus.
  • Normal level of prolactin is 10–25 ng/mL.
  • In microadenoma, it is between 100 ng/mL and 200 ng/mL.
  • In macroadenoma, it is greater than 200 ng/mL.
  • Immunoassay for prolactin does not reflect its bioassay levels always.
  • Many a times patients have no symptoms in spite of high prolactin level because of presence of macromolecules which are big prolactin molecules that account for 10–12% of hyperprolactin in a symptomatically normal patient.
Galactorrhea with Normal Prolactin Levels
Patients with galactorrhea present with normal serum prolactin levels. This may be mucoid discharge and not actual galactorrhea. Any discharge therefore from the breast should be seen under microscope. If it contains fat globules, only then it is galactorrhea.
When ovulation induction is done, in many patients estrogen leads to rise in prolactin levels in first half of the menstrual cycle. These patients have night spikes of high prolactin level and are known as spikers. This is transient hyperprolactinemia. The clinical presentation is poor endometrium in spite of good follicles. This condition is difficult to diagnose, but bromocriptine can be given in a dose of 1.25 mg twice a day, in these patients in first half of the cycle. It is a therapeutic test. If endometrium improves, it is continued in the subsequent cycle, otherwise it is stopped. Apart from bad oocyte quality, the spikers also have a high chance of LPD.
As discussed earlier, marginally high prolactin levels are also found in PCOS patients due to high estrogen and high DHEA-S. In these cases, bromocriptine or cabergoline is not required, PCOS is to be treated and this corrects hyperprolactinemia.
Detection of ovulation is extremely important to decide the further line of treatment in patients for treatment of infertility. There are various tests to detect ovulation and current status of each test is discussed here.
Clinical Presentation
  • Regular menstrual cycle (24–35 days) is in favor of ovulation. Any type of irregularity, beyond the limits (24–35 days), strongly indicates anovulation or dysovulation.16
  • “Mittelschmerz”: This is a mid-cycle pain in an ovulatory cycle because of contraction of smooth muscles surrounding the follicle about 12–24 hours prior to ovulation. This contraction helps the follicle to rupture and ovulation occurs.
  • Cervical mucus feel: Patient feels watery discharge at the introitus which becomes thin and copious prior to ovulation. This indicates a mature follicle and not ovulation.
  • Pregnancy is the only surest sign of ovulation.
  • Painful menstruation is also an indicator of the previous cycle being ovulatory.
Basal Body Temperature
The principle of basal body temperature (BBT) measurement is that progesterone secretion after ovulation raises the body temperature. BBT is between 97°F and 98°F during the follicular phase. After ovulation, it starts rising and goes high by 0.4–0.8°F, as the progesterone rises. BBT is at its lowest just before ovulation which is known as nadir. The temperature falls before the period or when progesterone level decreases due to regressing corpus luteum. The rise in temperature persists, in case the patient conceives and patient feels her body temperature as much as low-grade fever. This is because of persistent and high levels of progesterone from the corpus luteum of pregnancy, stimulated by hCG.
Basal body temperature is taken before rising from bed, with glass thermometer placed in oral cavity. The calibration on thermometer is 96–100°F. The change pattern in thermometer is known as biphasic pattern. The next period in normal cycle starts after 12 days of rise in temperature if pregnancy does not occur. It is a low cost, noninvasive and simple method and indicates abnormalities of follicular and luteal phase. But the temperature may be altered with smoking and disturbed sleep. It may be stressful for the patient to take temperature daily. There may not be shift in temperature in spite of ovulation and in this case, only after a few days of ovulation it may be diagnosed. This is not clinically useful. BBT is not commonly used in clinical practice.
Midcycle LH Surge Diagnosed by LH Kit
Luteinizing hormone surge occurs for 48–50 hours. LH has short half-life. LH rise can be detected by urinary LH kits. When the threshold level of 40 mIU/mL is crossed, test becomes positive on the kit and the color is seen. The intensity of the color increases as LH level increases. LH level normally starts rising early in the morning and may be misinterpreted as negative if urine is tested in the morning as it appears in the urine after 6–8 hours. Therefore, LH assessment in the afternoon is more reliable.
The test is done daily. But doing it twice daily decreases the false negatives. Too much of fluid intake is avoided few hours before the test to prevent dilution of the urine and LH in urine.
Ovulation occurs 20 hours ±6 hours after detection of urinary LH surge.4447 Patient is advised intercourse for 2 days following detection of LH surge as these days are the most fertile ones. If artificial insemination of donor (AID) is planned, it should also be done for 2 days. The specificity and sensitivity for LH surge are 90% and 96% respectively and if test is done twice a day, sensitivity reaches to 99%.47
So LH detection kits are noninvasive, self-monitored and indicate fertile period, 17so it is useful clinically for planning timed intercourse, IUI and AID. It can diagnose abnormality of follicular phase and luteal phase. But the disadvantage is that it is tedious, may be false negative and restricts patients from fluid intake. False positive results may occur with OC pills, clomiphene citrate, hCG, human menopausal gonadotropin (hMG) and danazol. But easy availability of ultrasound has reduced the utility of LH kits.
Endometrial Biopsy
It was used to document secretory changes in the endometrium after ovulation. Endometrial biopsy differentiates follicular phase from secretory phase. An experienced pathologist can date the endometrium after ovulation. Two days out of phase endometrium is regarded as LPD.48 This test is not as preferred as ultrasound or serum progesterone levels as it is inaccurate and highly invasive.
Luteal Serum Progesterone Levels
Progesterone level is below 1 ng/mL during follicular phase. Before ovulation it rises up to 1–2 ng/mL. The peak reaches after a week of ovulation. Level more than 3 ng/mL indicates ovulation. Normally on 7th postovulatory day, progesterone level is greater than 10 ng/mL.49 Serum progesterone level is only used to document ovulation as it is simple, fairly accurate, noninvasive and reliable.
Daily assessment of progesterone for diagnosis of LPD is not used as is inconclusive and multiple levels are required for accuracy.
Clinically estrogen peak will give copious mucus discharge and when dried on a slide will show a “fern” pattern. Before ovulation it shows tertiary branches. Normal preovulatory mucus strand can be stretched to 8–10 cm between two ends of artery forceps. Both these tests indicate high estrogen level, mostly due to mature follicle but do not indicate ovulation.
A follicle that is of greater than 10 mm in diameter, grows at a rate of 2–3 mm/day has no internal echogenicity and has thin (pencil line like) walls is not only more likely to become the leading follicle but will also give mature healthy ovum. The growing follicle can be assessed by transvaginal sonography. A mature follicle is 16–18 mm, has thin walls, regular round shape and no echogenicity in the lumen. When functionally mature, on color Doppler, the follicle shows blood vessels covering at least three-fourths of the follicular circumference (see Fig. 1.7). On pulse Doppler, these blood vessels show an RI of 0.4–0.4813 and PSV of greater than 10 cm/sec. The endometrium starts appearing multilayered on ultrasound as early as mid-proliferative phase with rising estrogen. On transvaginal scan, endometrial thickness of 6 mm is considered minimum that is required on the day of ovulation or on the day of hCG trigger for a successful outcome, although 8 mm is generally considered optimum.50 Segmental uterine artery perfusion demonstrates significant correlation with hormonal and histological markers of uterine receptivity, reaching the highest sensitivity for subendometrial blood flow.51
Endometrial vessels on pulse Doppler if have RI of less than 0.6, it have been reported to be a good prognostic factor for implantation.52 Rupture of the follicle is documented as disappearance of the follicle and it may be replaced by a cystic structure with thick shaggy walls and echogenicity in the lumen. But it is known to have variable appearances (Fig. 1.10) like ground glass echogenicity in lumen or lace-like echogenicities. This structure is known as corpus luteum.18
Corpus luteum can be seen as low resistance vascular structure with thick walls and internal echogenicities (Fig. 1.11).
Secretory changes are seen in the endometrium in the form of echogenicity of the endometrium which starts from outside in the early luteal phase, proceeding to the central line making a ring sign of the endometrium.53 Posterior wall of the uterus also appears more echogenic in this phase due to acoustic enhancement by the endometrium due to fluid accumulation (Fig. 1.12).
Serial ultrasound for documenting ovulation should not be done if intervention like ovulation trigger or IUI is not planned. Whether ovulation has occurred or not can be known by postovulatory serum progesterone level only.
Summarizing Diagnosis of Ovulation
  • Clinical history suggests ovulation in most cases.
  • Basal body temperature is inconclusive and not used routinely.
  • Urinary LH kit is useful only for patients who cannot visit clinic off and on. It is not very commonly used.
  • Serum progesterone greater than 3 ng/mL postovulatory indicates ovulation and is a most reliable means to document ovulation.
  • Daily ultrasound for just confirming that ovulation has occurred or not is not required unless some intervention is intended.
  • Endometrial biopsy is not required to assess the secretory phase changes.
  • Fern pattern and spinnbarkeit test indicate high estrogen level and not ovulation.
zoom view
Fig. 1.10: Corpus luteum on B-mode ultrasound.
zoom view
Fig. 1.11: Low resistance pericorpus luteal flow as seen on Doppler.
The incidence of infertility is 15–17% after 1 year of unprotected intercourse. After 2nd year, only 7–8% of couples remain infertile. After 2.5 years almost 7% will still remain infertile. Out of these infertile couples, in 20–40%, anovulation or oligo-ovulation is the causative factor.
The classification of causes of anovulation is very well described by the World Health Organization (WHO). It is classified as:
  • WHO group I: Hypothalamic-pituitary failure
  • WHO group II: Hypothalamic-pituitary dysfunction
  • WHO group III: Ovarian failure
  • WHO group IV: Hyperprolactinemia
  • WHO group V: Outflow defects (not associated with anovulation).
Hypothalamic-Pituitary Failure (WHO Group I)
It is also known as hypogonadotropic hypogonadism.
zoom view
Fig. 1.12: Ring sign of secretory endometrium as seen on B-mode ultrasound.
zoom view
Fig. 1.13: Amenorrhea/anovulation.
20In this situation, gonadotropin (FSH and LH) concentrations are so low that follicle is not stimulated and therefore there is extremely low estrogen level. These patients have anovulation and amenorrhea.
Hypothalamic Causes
  • Anorexia nervosa: It is weight related. It may be due to crash diet or frank anorexia nervosa.
  • Stress: It may be stress-related anovulation and amenorrhea. Severe exercises like marathon running can cause amenorrhea.
  • Kallmann syndrome: It is associated with anosmia.
  • Craniopharyngioma.
  • Debilitating systemic diseases.
  • Idiopathic.
Pituitary Causes
  • Hypophysectomy.
  • Severe postpartum hemorrhage (PPH) causing Sheehan's syndrome.
  • Radiotherapy for pituitary tumors.
In these conditions, GnRH replacement therapy is very useful. But gonadotropins are usually given for ovulation induction.
Hypothalamic-Pituitary Dysfunction (WHO Group II)
As contrast to WHO group I, in patients of this group, FSH is present that stimulates follicle and so estrogen is also present. Clinically this condition presents as oligo-ovulation or anovulation causing amenorrhea. As endogenous estrogen is present in these patients, 5 days of oral progesterone (10 mg hydroxyprogesterone acetate daily) will give withdrawal bleeding usually within 10 days and this is confirmatory of WHO type II anovulation. PCOS is the most common condition amongst WHO group II. Diagnosis of PCOS is made by Rotterdam criteria (2003). These are as follows:
  • Oligo-ovulation/anovulation
  • Clinical and/or biochemical hyperandrogenism
  • Polycystic ovaries on ultrasound.
Any two of these above mentioned three criteria establishes the diagnosis of PCOS. These criteria are defined as mentioned below:
  • Oligo-ovulation/anovulation: Oligo-ovulation is ovulation occurring once in 35–180 days. Anovulation is defined as no ovulation in 6 consecutive months.
  • Hyperandrogenism: clinical or biochemical: Clinical signs include hirsutism, acne, alopecia (male type) and female virilization. Biochemical indicators include raised total testosterone, androstenedione and free androgen index.
  • Polycystic ovaries on ultrasound: Presence of 12 or more follicles in either ovary, 2–9 mm in size and/or ovarian volume greater than 10 cc. The new cut off for follicle number is 20 per ovary.
Though the concepts on the number of follicles and ovarian volume for the diagnosis of PCOS are changing. An average value of 26 or more follicles per ovary is a reliable threshold for detecting polycystic ovaries in women with frank manifestation of PCOS. Sensitivity and specificity for diagnosis of PCOS for follicle number per ovary (FNPO) (26) were 85% and 94% and for ovarian volume (10 cc) were 81% and 84%. But the same study has also quoted that the lower follicle threshold may be required to detect milder variants of the syndrome.54
Hyperinsulinemia and high androgen levels are very useful for diagnosis. Postprandial insulin levels (2 hours after 75 g of glucose) are increased in patients with insulin resistance that is one of the major diagnostic features for PCOS. Along with this, increased stromal volume on 3D ultrasound is diagnostic of PCOS. Management includes lifestyle changes, 21oral ovulogens, gonadotropins and ovarian drilling.
Ovarian Failure (WHO Group III)
This group presents with high FSH along with low estrogen and amenorrhea. The patient will have symptoms of hypoestrinism. Physiologically this condition is found with the onset of menopause. But menopause may set in prematurely.
Causes of Ovarian Failure
  • Menopause (>40 years of age)
  • Premature menopause (<40 years of age):
    • Familial tendency
    • Autoimmune disease
    • Chemotherapy
    • Radiotherapy
    • Infections like tuberculosis
    • Idiopathic.
  • Chromosomal—Turner's syndrome.
Diagnosis is confirmed by high FSH greater than 25 IU/mL with extremely low AMH. The only available treatment option to achieve a pregnancy in these patients is ovum donation. Hormone replacement therapy (HRT) is required in these patients, to support their physiological needs.
Hyperprolactinemia (WHO Group IV)
Anovulation because of hyperprolactinemia is present when prolactin levels are double the upper normal limits. This leads to anovulation. Marginally high prolactin levels do not require any treatment as it does not lead to anovulation. Mildly high prolactin levels are found in PCOS but in these cases no therapy is required to decrease prolactin levels. Prolactin is regulated by “prolactin-inhibiting factor”, dopamine. Therefore, any hypothalamic lesion or drugs suppressing hypothalamic activity will increase prolactin level and LH will be low.
Outflow Defects (WHO Group V)
  • Primary: Imperforate hymen, absent uterus.
  • Secondary: Asherman's syndrome.
So chiefly three tests: (1) progesterone withdrawal bleeding, (2) serum prolactin and (3) serum FSH level assessment can give accurate diagnosis of anovulation for the management.
  • In all patients of anovulation/amenorrhea, always assess the prolactin levels as the first investigation to exclude hyperprolactinemia. (WHO IV)
  • Progesterone withdrawal bleeding indicates presence of endogenous estrogen and diagnosis of hypothalamic-pituitary dysfunction. (WHO II)
  • Low FSH indicates hypogonadotropic hypogonadism. (WHO I)
  • High FSH indicates ovarian failure. (WHO III)
  • Normal FSH with no withdrawal bleed indicates absent uterus or outflow tract obstruction or Asherman's syndrome. (WHO V)
  • Clinical examination:
    • Patient's height and weight are assessed for calculation of body mass index (BMI). (BMI = weight in kg/height2 in meters.) Normal BMI is 20–25, less than 20 is considered underweight and more than 30 is considered frank obesity. These values are decided according to the western standards, but may differ marginally for the Indian standards. Anovulation is common in patients with abnormal BMI.
    • With obesity, PCOS must be considered, which is characterized by hirsutism and/or acne, acanthosis nigricans, dark 22discoloration of skin in axilla and neck and waist circumference more than 35 inches or 90 cm. But it is important to remember here that these features will be seen only in full blown PCOS and there is a much larger percentage of infertile population that has silent forms or milder forms of PCOS.
    • Body mass index less than 20 may be seen in thin lean PCOS, malnourishment or anorexia nervosa. Causes of primary amenorrhea and ovarian failures must also be ruled out by excluding the clinical presentation of estrogen deficiencies.
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