Journal Pre-proofs Short communication A novel splice site variant in ANOS1 gene leads to Kallmann syndrome in three siblings Xiaohui Jiang, Dingming Li, Yanzi Gao, Xueguang Zhang, Xiang Wang, Yihong Yang, Ying Shen PII: DOI: Reference:
S0378-1119(19)30836-4 https://doi.org/10.1016/j.gene.2019.144177 GENE 144177
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
28 August 2019 16 October 2019 16 October 2019
Please cite this article as: X. Jiang, D. Li, Y. Gao, X. Zhang, X. Wang, Y. Yang, Y. Shen, A novel splice site variant in ANOS1 gene leads to Kallmann syndrome in three siblings, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene. 2019.144177
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Elsevier B.V. All rights reserved.
1
1
A novel splice site variant in ANOS1 gene leads to Kallmann
2
syndrome in three siblings
3
Xiaohui Jianga,b,1, Dingming Lia,b,1, Yanzi Gaoc, Xueguang Zhangd, Xiang Wangd,
4
Yihong Yange, Ying Shend,*
5
aHuman
6
Chengdu 610041, China
7
bKey
8
(Sichuan University), Ministry of Education, Chengdu 610041, China
9
cWest
Sperm Bank, West China Second University Hospital, Sichuan University,
Laboratory of Birth Defects and Related Disease of Women and Children
China School of stomatology, Sichuan University, Chengdu, 610041, China
10
dDepartment
11
(SCU-CUHK), Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases
12
and Birth Defects of Ministry of Education, West China Second University Hospital,
13
Sichuan University, Chengdu 610041, China
14
eCenter
15
University, Chengdu 610041, China
16
*Corresponding
17
*Correspondence address: Tel.: +86-15982083665; E-mail:
[email protected]
18
(Y. Shen)
19
1The authors consider that the first two authors should be regarded as joint First Authors.
20 21 22
of Obstetrics/Gynecology, Joint Laboratory of Reproductive Medicine
of Reproductive Medicine, West China Second University Hospital, Sichuan
author.
2
23
Highlight
24
A novel mutation in ANOS1 gene was identified in a Kallmann syndrome family.
25
This splice site variant results in a truncated protein due to a frameshift.
26
Timely hormone replacement therapy of KS depends largely on precise genetic
27
diagnosis.
28 29 30
Abstract
31
Idiopathic hypogonadotropic hypogonadism (IHH) is a rare genetic disease caused
32
by low doses of hypothalamic gonadotropin-releasing hormone (GnRH), leading to
33
absence or delayed sexual development. Kallmann syndrome (KS) is characterized by
34
IHH with anosmia or hyposmia. Here, we identified a novel splice site variant (c.
35
726+2T>G) of ANOS1 gene in three siblings with KS from a Chinese Han family by
36
whole-exome sequencing (WES). In this family, KS is classified as an X-linked
37
recessive inheritance pattern. This mutation was inherited from the mother by Sanger
38
sequencing. An in vitro functional experiment has identified the deleterious effect of
39
this mutation on the transcriptional level of ANOS1 gene. Importantly, the effectiveness
40
of timely hormone replacement therapy was evaluated on the three siblings. Hence,
41
finding genetic causes could be helpful in the early diagnosis and timely treatment of
42
KS.
43 44 45
Abbreviations:
3
46 47 48 49 50 51
IHH, Idiopathic hypogonadotropic hypogonadism; HPG, hypothalamo-pituitarygonadal; KS, Kallmann syndrome; WES, whole-exome sequencing; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; nIHH, normosmic IHH; CDGP, constitutional delay of growth and puberty; WAP, whey acidic protein; FnIII, fibronectin type III; HCG, human chorionic gonadotrophin; HMG, human menopausal gonadotrophin
52 53 54 55 56 57
Key words: idiopathic hypogonadotropic hypogonadism, Kallmann syndrome, ANOS1
58
gene, whole exome sequencing, mutation
59 60
1. Introduction
61
Idiopathic hypogonadotropic hypogonadism (IHH) is a heterogeneous disorder
62
characterized by delayed or absent puberty and infertility and is often accompanied by
63
other developmental abnormities, such as renal agenesis or hypoplasia, cleft lip/palate,
64
dental agenesis, ear anomalies, hearing impairment and bimanual synkinesis or skeletal
65
anomalies (Quinton, et al., 2001). The causative factors of IHH are complicated.
66
Recently, it was reported that known genetic defects account for 50% of all IHH
67
patients (Crowley, et al., 2008). To date, approximately 50 genes are associated with
68
IHH (Topaloglu, 2017). IHH can be divided into IHH with anosmia/hyposmia
69
(Kallmann syndrome [KS]) and normosmic IHH (nIHH). In addition, KS accounts for
70
half of all IHH cases (Boehm, et al., 2015). The reason that KS patients suffer from
4
71
anosmia/hyposmia is the abnormal migration of GnRH-specific neurons from their
72
origin in the olfactory placode to the forebrain (Schwanzel-Fukuda, et al., 1989;
73
Teixeira, et al., 2010). Therefore, loss-of-function mutations in genes related to GnRH
74
neuron migration will lead to KS. The major genes and their relative genetic models
75
involved in KS include ANOS1 gene (KAL1) in the X-linked form; FGFR1, FGF8,
76
WDR11, SOX10 and CHD7 in the autosomal-dominant form; and PROK2 and PROKR2
77
in the autosomal-recessive form (Boehm, et al., 2015). Among these genes, ANOS1
78
gene, is the second most commonly mutated gene, accounting for approximately 10–
79
20% of familial and sporadic KS patients. Currently, approximately 70 mutations in
80
ANOS1 gene have been identified, all of which are spread throughout the entire gene,
81
and no mutation “hot spots” have been found in the affected regions (Nie, et al., 2017).
82
The diagnosis of IHH is very important because hormone therapy is effective in
83
many IHH patients. Consequently, with the exception of typical manifestations, we
84
should also pay attention to genetic factors to facilitate IHH diagnosis because it is often
85
difficult to distinguish IHH from other similar diseases, such as the constitutional delay
86
of growth and puberty (CDGP) (Boehm, et al., 2015).
87
In this study, we report a novel splice site variant in ANOS1 gene leading to KS in
88
three siblings of a family. The results of functional experiments in vitro show that this
89
mutation results in ANOS1 protein truncation and further confirms the pathogenicity of
90
this variant. Thus, genetic testing is a good method to diagnose IHH, and effective
91
treatment can be provided in a timely manner.
92
5
93
2. Materials and methods
94
2.1 Study Participants
95
We describe three subjects in this study. The three patients are from one family and
96
diagnosed with infertility at Human Sperm Bank of West China Second University
97
Hospital. This study was approved by the Ethical Review Board of West China Second
98
University Hospital, Sichuan University (Project number: 2019019). Informed consent
99
was obtained from each subject in our study.
100 101
2.2 Whole-exome sequencing (WES) and Sanger sequencing
102
Blood samples were obtained from this family, and genomic DNA was extracted
103
using a whole blood DNA purification kit (QIAGEN, Germany). Whole-exome
104
sequencing (WES) was performed on the three patients. The candidate causative
105
mutation found by WES was confirmed in this family through Sanger sequencing. In
106
addition, mutation detection in 200 normal controls was performed by Sanger
107
sequencing analysis. The primers are: F 5'
108
R 5'
CTATTTTGAGGCATAGCAAGT
TCAGATGTGAAACCCTGTATG
3';
3'.
109 110
2.3 Minigene assay
111
A functional splicing reporter minigene assay was used to assess the impact of
112
sequence variants on splicing. A genomic segment encompassing exon 5, intron 5 and
113
exon 6 of ANOS1 gene was PCR-amplified from patient genomic DNA as well as the
114
normal control and was cloned into the minigene vector pSPL3. After transient
6
115
transfection into cultured cells, the splicing patterns of the transcripts generated from
116
the wild-type and variant constructs were compared by RT-PCR analysis and
117
sequencing.
118
ACGGGATCACCAGAATTCTGGAGCTCTTTGCTTTGCAGTGTTTTCC 3'; R 5'
119
GGCCCAAACATTATGTACCTCTGTATCATATGGCAAAGCCACCTGTTGACT
120
A 3'. The primers for RT-PCR are: SD6(F) 5' TCTGAGTCACCTGGACAACC 3';
121
SA(R) 5' ATCTCAGTGGTATTTGTCAGC 3'.
The
primers
for
genomic
amplification
are:
F
5'
122 123
3. Results
124
3.1 Case description
125
We report a non-consanguineous Chinese Han family that has six children, three of
126
whom are infertile (III-3, III-6, III-7) (Fig. 1). To identify the cause of the sterility of
127
the three patients, we carried out several related clinical examinations on them. The
128
karyotypes of the three siblings were 46, XY. However, the sex hormone examination
129
indicated that testosterone, FSH, and LH were all decreased. In addition, they showed
130
low peak LH and FSH responses after GnRH stimulation (Brito, et al., 1999). Moreover,
131
the three patients presented with smooth skin and a thin voice without facial hair or an
132
Adam's apple (Fig. 2). According to Tanner stages, all the testicles of the three siblings
133
were PH1G2, presenting as a small penis; bilateral testicles of III-3 and III-7 were 2 ml;
134
the left side of III-6 was 2 ml, and the right side had cryptorchidism (Fig. 2). Thus, we
135
initially diagnosed the three siblings with IHH depending on their low sex hormone
136
levels and poor development of secondary sexual characteristics. We further performed
7
137
a T&T olfactometer test on them, and the results of the test indicated that the three
138
siblings had varying degrees of olfactory abnormalities. The brain MRI of III-6 showed
139
a pituitary that was small and thin, and the other two brothers were normal (Fig. 2). The
140
details of the clinical features of the three siblings are shown in Table 1. There was no
141
phenotypic abnormality discovered in the other siblings or their parents.
142 143
3.2 Molecular genetic analysis
144
Approximately 50% of all IHH cases are caused by genetic defects (Crowley, et al.,
145
2008). To elucidate the underlying genetic cause of the three siblings, we performed
146
WES on them. Additionally, the pedigree analysis revealed an X-linked recessive
147
inheritance in this family (Fig. 1). Thus, we focused on homozygous mutations in these
148
patients. Briefly, variants were considered if (1) the minor allele frequency was <1% in
149
any database, including the gnomAD, ExAC Browser and 1000 Genomes Project; (2)
150
the variant affected coding regions or canonical splice sites; and (3) the variant was
151
predicted as damaging by PolyPhen-2, SIFT and MutationTaster tools. Consequently,
152
a novel deleterious hemizygous splice site variant (c. 726+2T>G) was identified in the
153
ANOS1 gene on the X chromosome, which is a definitely causative gene associated
154
with KS.
155
To confirm the putative contribution of this mutation, we investigated this variant
156
site by Sanger sequencing in the whole family (Fig. 3). The unaffected mother (II-2)
157
and one sister (III-2) carried a heterozygous mutation. The unaffected father (II-1) and
158
the other two sisters (III-5, III-8) did not have this mutation. Furthermore, we did not
8
159
detect this mutation in a sample of 200 Chinese Han controls, supporting the
160
pathogenicity of this variant. Thus, we conclude that this is a pathogenic variant
161
according to the American College of Medical Genetics guidelines leading to KS in this
162
family.
163 164
3.3 Mutation effect analysis
165
By bioinformatics analysis, we predicted that this splice site variant (c. 726+2T>G)
166
was likely to damage splicing and lead to abnormal protein expression. Unfortunately,
167
the ANOS1 gene is hardly expressed in blood. Therefore, we could not check the
168
damage using the patients’ blood samples. To this end, we carried out a functional
169
splicing reporter minigene assay to uncover the harmful effect of this mutation on
170
ANOS1 gene splicing. As shown in Fig. 4A, the RT-PCR product of the variant was
171
longer than that of the wild-type. Sequence analysis revealed that the variant caused 38
172
nucleotides of intron 5 to be retained between exon 5 and exon 6, which was expected
173
to result in a truncated protein due to a frameshift and premature termination codon (p.
174
Thr243Glyfs*8) (Fig. 4B). Thus, the splice site mutation in intron 5 was the genetic
175
cause of KS in this family.
176 177
4. Discussion
178
IHH is a rare genetic disorder with a prevalence of 1:30,000 in males and 1:125,000
179
in females, which mainly impairs sexual development in puberty and subsequently
180
results in infertility in adults (Laitinen, et al., 2011). Therefore, it is necessary to provide
9
181
an effective treatment for IHH patients during puberty or preadolescence, which
182
depends on the accurate and efficient diagnosis of IHH. Hitherto, the diagnosis of IHH
183
has been based on clinical, biochemical, and imaging examinations. However, it is
184
particularly challenging to differentiate IHH and CDGP in early adolescence. Therefore,
185
genetic testing is a good way to diagnose IHH. By next-generation sequencing, nearly
186
50 genes have been identified to be related to IHH (Boehm, et al., 2015). Here, by WES,
187
we detected a novel intronic ANOS1 gene mutation in three siblings with KS, which
188
changes the splice site and forms a truncated protein.
189
KS is a form of IHH with anosmia or hyposmia. Mutations in genes regulating
190
GnRH neuron development, migration, and function are the key causative factors
191
(Topaloglu and Kotan, 2016). Additionally, ANOS1 gene is the first described
192
pathogenic gene associated with KS, and mutations are low in sporadic KS patients but
193
much higher in familial KS patients (Hamada, et al., 2013). ANOS1 gene is located on
194
the X chromosome and encodes an extracellular glycoprotein called Anosmin-1, which
195
contains four domains: an N-terminal cysteine-rich (Cys-box) domain, a whey acidic
196
protein (WAP) domain, four fibronectin type III (FnIII) domains, and a histidine-rich
197
C terminal region (del Castillo, et al., 1992). Anosmin-1 plays several roles in the
198
development of the central nervous system: promoting neuronal cell adhesion, neurite
199
outgrowth, axonal guidance, CNS projection neuron branching, and the migration of
200
multiple types of neuronal precursors, including GnRH-producing neurons and
201
oligodendrocyte precursors (Nie, et al., 2017). Mutations in ANOS1 gene disrupt the
202
protein structure and impair its functions, thus leading to KS (Hu, et al., 2003). In our
10
203
study, the splice site variant (c. 726+2T>G) causes a truncated ANOS1 protein and
204
destroys the FnIII domain (Fig. 5), which is involved in cell adhesion, tyrosine kinases
205
and phosphatases, which are implicated in neuronal migration and in axon guidance
206
(Legouis, et al., 1993). Therefore, dysfunctional Anosmin-1 is unable to mediate the
207
normal function of GnRH neurons and causes a defect in GnRH production, secretion,
208
or action, consequently resulting in KS.
209
On the Y chromosome, there is a pseudogene of ANOS1 gene named ANOS2P.
210
Although ANOS2P could not produce a functional protein, the sequences of ANOS1
211
gene and ANOS2P have a high degree of similarity, ranging from 86.2%-98.3% for
212
exons and from 86.3%-99.1% for introns (del Castillo, et al., 1992). Accordingly, when
213
screening the ANOS1 gene variants in male IHH patients through PCR sequencing, the
214
pseudogene ANOS2P should not be amplified. Herein, we designed primers located in
215
the region where the two genes are different to validate the splice site variant in this
216
family by PCR sequencing. The results of PCR sequencing are consistent with WES
217
results.
218
In approximately 10% of KS patients, hormone levels, testis or penile volumes
219
recover or grow spontaneously (Topaloglu and Kotan, 2016). Most cases of IHH are
220
caused by a spectrum of abnormal GnRH secretory patterns, including deficient
221
synthesis or secretion of endogenous GnRH (Spratt, et al., 1987), and so it is very
222
responsive to hormonal therapy. Consequently, for the remaining patients, hormone
223
replacement therapy is extremely essential. Approximately 92% of IHH patients can
224
reach normal adult testicular volume and have sperm production after treatment with a
11
225
GnRH pituitary pump (Pitteloud, et al., 2002). In the present study, the GnRH
226
stimulation test showed that the pituitary glands of the three siblings responded to
227
exogenous GnRH, which provided a laboratory basis for subsequent treatment and
228
prognosis. The three siblings were treated with 2000 U of human chorionic
229
gonadotrophin (HCG) and 75 U of human menopausal gonadotrophin (HMG) twice
230
weekly. Eight months later, the Tanner stage of the three patients changed from PH2G2
231
to PH2G3 or PH3G3. Specifically, the testicular size increased, and nocturnal penile
232
tumescence became frequent, and spermatorrhea was occasional. Taken together, our
233
results show that it is necessary to diagnose IHH and treat it in a timely manner, and
234
most IHH patients will have a good prognosis.
235 236
5. Conclusion
237
In conclusion, in this study, we identified a novel pathogenic mutation of ANOS1
238
gene in three siblings from a Chinese Han family, thus expanding the known spectrum
239
of ANOS1 gene mutations. The in vitro functional experiment elucidated the
240
pathogenesis of this mutation in KS. In this family, hormone replacement therapy
241
effectively induced sexual maturation. Therefore, genetic analysis plays a crucial role
242
in IHH diagnosis and prognosis and further provides beneficial knowledge related to
243
genetic counselling.
244 245
Acknowledgements
246
We thank all the patients who provided samples to this research. This work was
12
247
supported by General Program of China Post-doctoral Science Foundation
248
(2018M640920), Postdoctoral fund of Sichuan University (20826041B4090) and
249
Sichuan Science & Technology Program (2018SZ0144).
250 251
Authors’ roles
252
X.J. and D.L. collected blood samples and conducted the clinical evaluations. X.W.,
253
X.Z. and Y.Y. performed PCR, minigene assay and sanger sequencing analysis. Y.S.
254
designed this study and wrote manuscript.
255 256
Declaration of interest
257
The authors declare that they have no conflicts of interest with the contents of this
258
article.
259
260
261
262
263
264
265
Reference
266
Boehm U, Bouloux PM, Dattani MT, de Roux N, Dode C, Dunkel L, Dwyer AA,
13
267
Giacobini P, Hardelin JP, Juul A et al. Expert consensus document: European
268
Consensus Statement on congenital hypogonadotropic hypogonadism--pathogenesis,
269
diagnosis and treatment. Nat Rev Endocrinol 2015;11: 547-564.
270
Brito VN, Batista MC, Borges MF, Latronico AC, Kohek MB, Thirone AC, Jorge BH,
271
Arnhold IJ, Mendonca BB. Diagnostic value of fluorometric assays in the evaluation
272
of precocious puberty. J Clin Endocrinol Metab 1999;84: 3539-3544.
273 274
Crowley WF, Jr., Pitteloud N, Seminara S. New genes controlling human reproduction and how you find them. Trans Am Clin Climatol Assoc 2008;119: 29-37.
275
del Castillo I, Cohen-Salmon M, Blanchard S, Lutfalla G, Petit C. Structure of the X-
276
linked Kallmann syndrome gene and its homologous pseudogene on the Y
277
chromosome. Nat Genet 1992;2: 305-310.
278 279 280 281
Hamada AJ, Esteves SC, Agarwal A. A comprehensive review of genetics and genetic testing in azoospermia. Clinics (Sao Paulo) 2013;68 Suppl 1: 39-60. Hu Y, Tanriverdi F, MacColl GS, Bouloux PM. Kallmann's syndrome: molecular pathogenesis. Int J Biochem Cell Biol 2003;35: 1157-1162.
282
Laitinen EM, Vaaralahti K, Tommiska J, Eklund E, Tervaniemi M, Valanne L, Raivio
283
T. Incidence, phenotypic features and molecular genetics of Kallmann syndrome in
284
Finland. Orphanet J Rare Dis 2011;6: 41.
285
Legouis R, Lievre CA, Leibovici M, Lapointe F, Petit C. Expression of the KAL gene
286
in multiple neuronal sites during chicken development. Proc Natl Acad Sci U S A
287
1993;90: 2461-2465.
288
Nie M, Xu H, Chen R, Mao J, Wang X, Xiong S, Zheng J, Yu B, Cui M, Ma W et al.
14
289
Analysis of genetic and clinical characteristics of a Chinese Kallmann syndrome
290
cohort with ANOS1 mutations. Eur J Endocrinol 2017;177: 389-398.
291
Pitteloud N, Hayes FJ, Dwyer A, Boepple PA, Lee H, Crowley WF, Jr. Predictors of
292
outcome of long-term GnRH therapy in men with idiopathic hypogonadotropic
293
hypogonadism. J Clin Endocrinol Metab 2002;87: 4128-4136.
294
Quinton R, Duke VM, Robertson A, Kirk JM, Matfin G, de Zoysa PA, Azcona C,
295
MacColl GS, Jacobs HS, Conway GS et al. Idiopathic gonadotrophin deficiency:
296
genetic questions addressed through phenotypic characterization. Clin Endocrinol
297
(Oxf) 2001;55: 163-174.
298
Schwanzel-Fukuda M, Bick D, Pfaff DW. Luteinizing hormone-releasing hormone
299
(LHRH)-expressing cells do not migrate normally in an inherited hypogonadal
300
(Kallmann) syndrome. Brain Res Mol Brain Res 1989;6: 311-326.
301
Spratt DI, Carr DB, Merriam GR, Scully RE, Rao PN, Crowley WF, Jr. The spectrum
302
of abnormal patterns of gonadotropin-releasing hormone secretion in men with
303
idiopathic hypogonadotropic hypogonadism: clinical and laboratory correlations. J
304
Clin Endocrinol Metab 1987;64: 283-291.
305
Teixeira L, Guimiot F, Dode C, Fallet-Bianco C, Millar RP, Delezoide AL, Hardelin
306
JP. Defective migration of neuroendocrine GnRH cells in human arrhinencephalic
307
conditions. J Clin Invest 2010;120: 3668-3672.
308 309 310
Topaloglu AK. Update on the Genetics of Idiopathic Hypogonadotropic Hypogonadism. J Clin Res Pediatr Endocrinol 2017;9: 113-122. Topaloglu AK, Kotan LD. Genetics of Hypogonadotropic Hypogonadism. Endocr Dev
15
311
2016;29: 36-49.
312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
Figure 1. Family pedigree. Dark box: Infertile man (III-3, III-6 and III-7).
331
Figure 2. Phenotypes of three siblings with KS. All of the three patients with smooth
332
skin and barrenly facial hair or without an Adam's apple; the brain MRI showed III-6
16
333
presenting a small and thin pituitary (red arrow) and the pituitaries of III-3 and III-7
334
were normal (white arrows).
335
Figure 3. Sanger sequencing results of this family. The black arrows point to the
336
mutation site (c. 726+2T>G) in ANOS1 gene.
337
Figure 4. Functional effect of the splice site mutation on ANOS1 gene transcript. (A)
338
Agarose gel electrophoresis of RT-PCR fragments obtained from wild-type plasmid
339
and mutant plasmid. A measurable increase in molecular weight was observed in
340
mutant plasmid (4) compared with wild-type plasmid (3). A 100-bp ladder molecular-
341
weight marker (1) is shown. 2, blank control; 5, empty vector. (B) Sequence analysis
342
of the RT-PCR product obtained from mutant plasmid demonstrated the gain of 38
343
nucleotides from intron 5 between exon 5 and exon 6.
344
Figure 5. The domains of ANOS1 protein and the position of truncated protein. ANOS1
345
protein contains a WAP super family and three FN3 super family.
346 347 348 349 350 351
Table 1. Clinical and hormonal characteristics of patients in the family
Subject
III-7
III-6
Age(years) Gender Height/weight (cm/Kg)
21 Male 176/64
26 Male 179/70.5
Tanner stage
PH1G2
PH1G2
Testicular size (ml)
bilateral 2ml
Left 2ml, right cryptordism
17
Penis length
micropenis
micropenis
T&T olfactometer test
1.60
2.40
Karyotype
46, XY
46, XY
E2 (pg/ml)
11.8
34.0
T (ng/ml)
0.42
0.30
FSH (IU/L)
0.6
0.3
LH (IU/L)
0.1
0.4
LH (IU/L; basal/stimulated for 15 min/30min/60min)
0.2/0.7/1.1/1.0
0.1/0.65/1.3/1.7
FSH (IU/L; basal/stimulated for 15 min/30min/60min)
0.4/1.0/1.9/2.7
0.15/0.9/1.6/1.9
Basal hormone concentrations
GnRH excitatory experiment
352 353 354 355
E2, Estradiol; T, testosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GnRH, gonadotropin-releasing hormone.