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Diagnosis of 5[alpha]-reductase 2 deficiency: is measurement of dihydrotestosterone essential?

5[alpha]-Reductase 2 is a membrane-bound NADPH-dependent microsomal enzyme encoded by SRD5A2 [steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 2)]. The enzyme consists of 254 amino acids, catalyzing the reduction of the double bond between carbons 4 and 5 in the A ring in a variety of steroid substrates such as testosterone (T), [11] progesterone, and cortisol (1). Deficiency in this enzyme is characterized by undervirilization of the male external genitalia at birth due to failure to synthesize dihydrotestosterone (DHT), a hormone that is 10-fold more potent than testosterone in inducing signaling via the androgen receptor (1). Patients may present with almost complete female phenotype or ambiguous external genitalia, hypospadias, micropenis, bifid scrotum, single urogenital opening, or a combination of these defects. The diagnosis is made either in infancy or at puberty, when there is virilization of the external genitalia in female patients who have a karyotype of 46,XY.

Classic biochemical hallmarks of 5[alpha]-reductase 2 deficiency (5ARD) include a normal to high male concentration of serum T, low concentration of DHT, and increased T/DHT ratio at baseline or after human chorionic gonadotropin (hCG) stimulation. However, equivocal or false-negative T/DHT results are not uncommonly reported (2-6). Alternative approaches in diagnosing 5ARD include the use of urinary steroid profiling (USP) (7) and mutational analysis of SRD5A2 (8). USP has an established role in the diagnosis of various steroidogenesis defects owing to its characteristic steroid metabolite excretion pattern. Extremely low 5[alpha]-to 5[beta]-reduced steroid metabolite ratios are pathognomonic for 5ARD. On the other hand, the increasing availability of molecular diagnostic services in the 21st century is a promising alternative. Here, we share our experience in diagnosing 5ARD with the latter 2 approaches and propose new diagnostic algorithms for 46,XY disorders of sex development (DSD).

Materials and Methods

PATIENTS

We reviewed all the patients diagnosed with 5ARD by USP or mutational analysis of the SRD5A2 gene between January 2003 and July 2012 who were under the care of the departments of pediatrics and medicine in 6 public hospitals in Hong Kong. Clinical presentation, karyotype, and baseline and post-hCG stimulation T and DHT results were recorded. Except for patient 12, who is Indian, all patients are Chinese; none of their parents are consanguineous.

URINARY STEROID PROFILING

The Chemical Pathology Laboratory of the Queen Elizabeth Hospital is the only clinical laboratory in Hong Kong providing USP. Spot urine from patients <3 months of age and 24-h urine from those [greater than or equal to] 3 months of age were processed for steroid profiling as described previously (9). Briefly, the urine was extracted with Sep-Pak[R] cartridges (Waters), hydrolyzedby Helixpomatia digestive juice (BioSepra), reextracted on SepPak cartridges, and used to prepare methyl oximetrimethylsilyl derivatives. We analyzed the products using gas chromatography (model 6890N, Agilent Technologies) with a CP-Sil 5 capillary column (25 m x 0.32 mm, Varian) coupled to a mass spectrometer (model 5973, Agilent Technologies). The temperature program was as follows: injection at 106[degrees]C was held for 1 min, raised at 20[degrees]C/min to 180[degrees]C, increased to 275[degrees]C at 2.5[degrees]C/min, and held for 12 min. Data were acquired at 70 eV in cyclic scanning mode at a rate of 1.17 scans/s and mass range 50.00-700.00 amu. We quantified individual steroid metabolites by selected ion monitoring (9). Intra- and interassay imprecision ranges for various metabolites were 1.3%-11.9% and 1.3%-13.3%, respectively. The results were expressed in [micro]g/dL for spot urine and [micro]g/day for 24-h urine and were interpreted with reference intervals set up from age-matched local healthy subjects. Male reference intervals were considered the most appropriate for data interpretation in this study. The diagnosis of 5ARD is based on the finding of low ratios between 5[alpha]- and 5[beta]-reduced steroid metabolites, namely 5[alpha]-tetrahydrocortisol (5[alpha]-THF)/THF, androsterone (Ad)/aetiocholanolone (Ae), 11hydroxyandrosterone (11-OH Ad)/11-OH Ae, and 5[alpha]-tetrahydrocorticosterone (5[alpha]-THB)/THB. For newborns presenting with incomplete masculinization, this approach is not informative, since excretion of the THF and THB epimers (derived from cortisol and corticosterone, respectively) is negligible. At that period of life, the preferred catabolic route is via the 11-oxo equivalents, cortisone and 11-dehydrocorticoisterone. THF and THB epimers become reliably detectable from 3 months in term infants.

MUTATIONAL ANALYSIS OF SRD5A2

We performed mutational analysis of SRD5A2 after obtaining written consent from patients and/or parents and approval by the local clinical ethics committee. Genomic DNA was extracted from peripheral blood with a commercial kit (QIAamp DNA blood kit, Qiagen). All the coding regions and splice sites of SRD5A2 were amplified by PCR and sequenced as described previously (10). We used the reference genomic sequence NG_008365.1 and reference cDNA sequence NM_000348.3 for result interpretation.

IN SILICO ANALYSIS FOR NOVEL SPLICE-SITE MUTATION

The effect of a novel mutation detected at the splice site in 1 of the patients was predicted by the online in silico analysis software Fruitfly.org and NetGene2.

LITERATURE REVIEW

We performed a PubMed search with the keyword "5-[alpha] reductase deficiency" and the following filters: full text available, published in the last 10 years, humans, English language. Publications related to the investigation and diagnosis of 5ARD were reviewed in detail. We recorded the number of patients, concentration of DHT at baseline and after hCG stimulation, corresponding diagnostic cutoffs, methodology of the DHT assay, and findings of the mutational analysis of SRD5A2.

Results

Altogether, 16 patients were diagnosed with 5ARD in our laboratory in the past 10 years. Four of them had DHT measured by RIA in an overseas laboratory, with 2 reaching the diagnostic cutoff of 5ARD after hCG stimulation (patients 4 and 13). Fifteen patients underwent USP. All of them showed characteristic findings compatible with 5ARD (Tables 1 and 2). Because at <3 months of age, insignificant amounts of the 5[alpha]- and 5[beta]-reduced cortisol metabolites are excreted, patient 1 had the genetic test performed directly to facilitate early sex assignment. Except for patients 2, 3, 12, and 13, all patients underwent mutational analysis of SRD5A2, with 2 mutations detected to confirm the diagnosis. p.R227Q is the most common mutation detected in our patients, being homozygous in 4 patients and heterozygous in 4 others. A novel mutation, c.548-1G>A, was also detected in the heterozygous state in patient 14 (Fig. 1). In silico analysis showed that the natural splice site is abolished in the mutant sequence, suggesting that this novel variant is a pathogenic mutation.

We found 74 publications in the PubMed search under the conditions listed above, with 24 of them fulfilling the criteria for review (Table 3). In these 24 publications, clinical and laboratory data of 149 patients with 5ARD were reported. Sixty-nine patients (46.3%) had DHT measured at baseline, and 50 (33.6%) of them had DHT measured after hCG stimulation. Only 1 article included the diagnostic cutoffs for both baseline and post-hCG-stimulated T/DHT ratio. One mentioned the baseline and 2 mentioned the post-hCG-stimulated cutoffs they used for result interpretation. Two reports provided the cutoff but without specifying the time point. Eleven articles mentioned the methodology of their DHT assays. USP was used for diagnosing 5ARD in 3 studies, with all the patients showing low 5[alpha]- and 5[beta]-reduced steroid metabolite ratios (5, 10, 12). Regarding molecular diagnosis, 141 patients (94.6%) had 2 mutations detected in SRD5A2 to confirm the diagnosis. Six patients had only 1 mutation detected (2, 11, 13), and 2 patients had no mutation detected in the SRD5A2 gene (11).

[FIGURE 1 OMITTED]

Discussion

From the pathophysiological point of view, measuring the DHT concentration appears to play a central role in the diagnosis of 5ARD. An exaggerated T/DHT ratio has high positive predictive value and appears to be particularly useful in early infants (21). Yet, equivocal or false-negative results are also not uncommonly reported (2, 26). Most of the patients we report here did not rely on this test for diagnosis. Patient 3 had severely underdeveloped androgen-responsive tissues, and patients 13-16 had orchidectomy performed. Both of these circumstances may preclude meaningful application of the DHT test. An additional problem for us is that the DHT assay is not available in Hong Kong. Importing the authentic standards of DHT is 1 of our major hurdles in setting up mass spectrometry-based assays, since there are stringent customs regulations for importing anabolic steroids. The relatively infrequent request for this test inevitably decreases cost-effectiveness if RIA is used. On the other hand, transportation of human samples to overseas laboratories is expensive. Results obtained from different laboratories are difficult to interpret owing to heterogeneous assay platforms for T and DHT and the lack of unified cutoff for the T/DHT ratio. As seen in our patients, the DHT tests were performed by RIA in an overseas laboratory, whereas T was measured locally with various methodologies. All the above factors triggered us to revisit the role of DHT testing and look for alternative strategies for the diagnosis of 5ARD.

Reliable analytical methods and relevant reference intervals are both crucial to data interpretation. Although the analytical performance of the DHT assay has improved considerably with the migration from immunoassay-based to mass spectrometry-based technology (29), comprehensive study on the T/DHT reference values in different age-, sex-, and ethnic-specific groups is lacking. Kulle et al. (30) have published reference data on several androgens, including DHT, for pediatric age groups. Nevertheless, their sample size was far from ideal, with some reference data derived from as few as 4 individuals. Ethical concerns prevent collection of post-hCG-stimulated hormone data on healthy individuals. Furthermore, extensive validation of the pathologic cutoffs can hardly be achieved, because of heterogeneous patient data and the small number of patients under the care of each center. Of the 24 articles we reviewed, fewer than half provided the methodology of DHT measurement. A majority of the publications did not provide the cutoff they used for interpreting the T/DHT ratio. Including this information in all future publications would help share the experience of the diagnosis of this disease in a more comprehensive manner.

[FIGURE 2 OMITTED]

In view of the above limitations of the DHT test, we resorted to USP and genetic testing for the diagnosis of 5ARD, since both are readily available in our locality. Of the 15 patients undergoing USP, all showed low ratios in at least 2 of the 4 pairs of 5[alpha]-and 5[beta]-reduced steroid metabolites. The ratio of 5[alpha]-THF/THF is particularly useful, mainly because 5[alpha]-THF and THF are present at relatively higher concentrations in healthy individuals than the components of the other 3 pairs of metabolites. In patients with 5ARD, this ratio is particularly exaggerated, with most patients having a value close to 0. According to the reference intervals we derived from local healthy individuals, the sensitivity of this pair of metabolites is 100%. This is different from the observation of Berra et al. (12), who reported that the sensitivity of 5[alpha]-THF/THF was only 90% and that of Ad/Ae and 5[alpha]-THB/THB was 100%. Considering the results of all these pairs of metabolite ratios should help to diagnose all patients with 5ARD. Nonetheless, there are a few other conditions which may give similar findings, e.g., Cushing syndrome (31), hypothyroidism (32), anorexia nervosa (33), and consumption of some drugs (34). The ratios in these patients are seldom as extreme as in 5ARD. Taking into consideration the clinical context and the metabolite profile as a whole should avoid a false diagnosis of 5ARD.

Although the diagnosis of 5ARD by USP appears straightforward, caution must be exercised when interpreting results for neonates and young infants, since metabolites of cortisol are almost exclusively derived from the 11-oxo metabolite cortisone at this early-stage of life (35). An ongoing study in our laboratory shows that substantial amounts of 11-hydroxy cortisol metabolites, including THF and 5[alpha]-THF, start to appear only at around 3 months of age in healthy term infants (n = 20; unpublished data), whereas only trace amounts were detected in younger infants (n = 40; unpublished data). As a result, instead of USP, we propose using mutational analysis of SRD5A2 for making the diagnosis. The SRD5A2 gene consists of only 5 exons and 4 introns, which is technically easy to handle. The rate of mutation detection is encouraging, being 100% in our patients and close to 95% in reported cases in the past 10 years. With molecular diagnostic services becoming more widely available in most parts of the world, and with an undeniable advantage of making an early diagnosis to facilitate sex assignment and enable early use of topical DHT treatment for 5ARD patients (36), mutational analysis of the SRD5A2 gene should be considered a first-line test in the investigation of 46,XY DSD.

On the basis of the above evidence, we propose 2 algorithms to test for adrenal and gonadal function to differentiate between different etiologies of 46,XY DSD, taking into consideration test availability and the volume of blood required for the various investigations, with the latter being a particular concern for neonates and young infants. The test priority could be modified on the basis of clinical findings, and some tests could be performed simultaneously to minimize venipuncture and hasten the diagnosis. Neither of these algorithms (Fig. 2, A and B) includes DHT. Instead, mutational analysis of SRD5A2 is the key to the diagnosis of 5ARD. The diagnostic algorithm in Fig. 2A is suitable to those centers where USP is readily available. Some centers providing this test can accept samples sent by airmail in the form of extracts preserved on solid-phase extraction cartridges. Because USP provides diagnostic information on a number of relatively common causes of 46,XY DSD, it reduces the volume of blood required for other hormone testing. The algorithm in Fig. 2B is suitable to those centers where USP is not readily available. It involves a larger blood volume for the various laboratory investigations. In both algorithms, genetic testing should follow the biochemical phenotyping as far as possible to confirm the diagnosis and provide information on genetic counseling. Nonetheless, when DHT testing is performed, it still provides valuable clues to the differential diagnosis of 46,XY DSD, bearing in mind the limitations of the test as stated above.

Massively parallel sequencing technology represents a major breakthrough in clinical laboratory medicine in the last decade. Multiple genes can be sequenced in a single analysis, significantly reducing overall labor input and analytical time. Its high capacity may help uncover potentially novel pathophysiological pathways and the interactions of various genetic alterations in modifying phenotypic expression. Application of this technology for the genetic diagnosis of DSD has been published recently (37). It is hoped that when more experience on this technology is gathered and when its value in clinical application is thoroughly tested, most, if not all, of the patients with DSD will have a confirmed diagnosis.

The major limitation of this study is that not all patients gave consent for genetic testing after biochemical phenotyping with USP. As a result, phenotype-genotype correlation cannot be studied comprehensively. Nevertheless, for those with both tests performed, the concordance rate is 100%, providing strong evidence that USP is an ideal test for biochemical phenotyping in 5ARD after 3 months of age with the rationale stated above.

In conclusion, 5ARD can be confidently diagnosed by USP from 3 months postnatally and by mutational analysis of SRD5A2. DHT testing is not widely available and its result interpretation maybe problematic. Its role in the diagnosis of 5ARD has been overemphasized.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: No sponsor was declared.

Acknowledgments: We thank Dr. Norman F. Taylor for his critical comment on the manuscript, and M.M. Lau, C.W. Cheng, W.K. Hui, W.T. Lo, M.S. Ma, and Y.F. Wong for their technical assistance in performing the laboratory tests.

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Angel On Kei Chan, [1] * Betty Wai Man But, [2] Ching Yin Lee, [3] Yuen Yu Lam, [4] Kwok Leung Ng, [5] Joanna Yuet Ling Tung, [6] Elaine Yin Wah Kwan, [7] Yuk Kit Chan, [8] Teresa Kam Chi Tsui, [9] Almen Lai Na Lam, [5] Wing Yee Tse, [2] Pik To Cheung, [6] and Chi Chung Shek [10]

[1] Division of Clinical Biochemistry and [6] Department of Paediatrics, Queen Mary Hospital, Hong Kong SAR; [2] Department of Paediatrics and [10] Department of Pathology, Queen Elizabeth Hospital, Hong Kong SAR; [3] Department of Paediatrics and [8] Department of Medicine, Caritas Medical Centre, Hong Kong SAR; [4] Department of Paediatrics, Kwong Wah Hospital, Hong Kong SAR; [5] Department of Paediatrics, United Christian Hospital, Hong Kong SAR; [7] Department of Paediatrics, Pamela Youde Nethersole Eastern Hospital, Hong Kong SAR; [9] Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong SAR.

* Address correspondence to this author at: Division of Clinical Biochemistry, Department of Pathology and Clinical Biochemistry, Queen Mary Hospital, 102 Pok Fu Lam Road, Hong Kong SAR. Fax ?28559155; e-mail: cok436@ha.org.hk.

Received September 18, 2012; accepted February 5, 2013.

Previously published online at DOI: 10.1373/clinchem.2012.196501

[11] Nonstandard abbreviations: T, testosterone; DHT, dihydrotestosterone; 5ARD, 5[alpha]-reductase 2 deficiency; hCG, human chorionic gonadotropin; USP, urinary steroid profiling; DSD, disorders of sex development; THF, tetrahydrocortisol; Ad, androsterone; Ae, aetiocholanolone; 11-OH Ad, 11-hydroxyandrosterone; THB, tetrahydrocorticosterone.
Table 1. Findings in urinary steroid profiling and mutational analysis
of SRD5A2 in 46,XY patients with 5ARD in Hong Kong. (a)

          Sex of    Presenting          Age when USP    5[alpha]-
Patient   rearing   features            performed        THF/THF

1            M      Hypospadias,        5 days             ND (c)
                      bifid
                      scrotum,
                      micropenis

2            M      Micropenis          6 months          0.05

3            M      Micropenis,         10 months         0.03
                      hypoplastic
                      scrotum

4            M      Micropenis          3 years           0.02

5            M      Micropenis,         3 years           0.04
                      right
                      undescended
                      testis
6 (g)        M      Hypospadia          4 years           0.00
7 (g)        M      Micropenis          5 years           0.03
8            M      Micropenis          6 years           0.08
9 (g)        M      Micropenis          7 years           0.05
10 (g)       M      Micropenis          8 years           0.02
11 (g)       M      Micropenis          17 years          0.02
12           M      Hypospadias,        25 years          0.04
                      bifid scrotum,
                      micropenis
13           F      Ambiguous           2 years           0.03
                      genitalia
                      at birth
14           F      Ambiguous           19 years          0.04
                      genitalia
                      at birth
15           F      Ambiguous           25 years          0.02
                      genitalia
                      at birth
16 (g)       F      Ambiguous           26 years          0.03
                      genitalia
                      at birth

                          11-OH
                         Ad/11-         5[alpha]-
Patient   Ad/Ae           OH Ae          THB/THB

1         ND               ND              ND
2         1.00 (d)      3.00 (d)          0.28
3         1.50 (d)        2.00            0.33
4         0.29            0.67            0.33
5         0.24            2.79            0.20
6 (g)     0.11            0.90            0.20
7 (g)     0.21            0.07            0.20
8         0.10            0.18            0.02
9 (g)     0.19            0.50            0.22
10 (g)    0.05            0.14            0.21
11 (g)    0.23            1.85            0.27
12        0.15            0.18            0.19
13        0.55            1.43            0.43
14        0.35            0.73            0.36
15        0.07            0.37            0.08
16 (g)    0.19          1.68 (d)          0.22

                            Baseline

Patient      T, ng/dL       DHT, ng/dL       T/DHT

1              77.8            <6.1          >12.8
2              184.4           11.0          16.8
3              <4.9            <4.9           NA
4            141.2 (e)          ND            NA
5              <11.5            ND            NA
6 (g)          86.5             ND            NA
7 (g)        <11.5 (h)          ND            NA
8            <11.5 (i)          ND            NA
9 (g)         196 (j)           ND            NA
10 (g)          ND              ND            NA
11 (g)         412.1            ND            NA
12             89.3             ND            NA
13              4.9             1.7           1.7
14              ND              ND            NA
15              ND              ND            NA
16 (g)       123.9 (k)          ND            NA

                 After hCG stimulation

Patient   T, ng/dL   DHT, ng/dL   T/DHT     SRD5A2 genotype
                                   (b)

1          478.4        54.1       8.8      p.N193S/p.N193S
2            ND          ND         NA             ND
3          167.1         11        15.2            ND
4          403.5f      20.1f       20.1     p.R227Q/p.R227Q
5          233.4         ND         NA       p.Q6X/p.R227Q
6 (g)      288.2         ND         NA       p.V10G/p.R227Q
7 (g)        ND          ND         NA       p.Q6X/p.R227Q
8          100.9         ND         NA      p.R227Q/p.R227Q
9 (g)      213.3         ND         NA       p.L55P/p.R227Q
10 (g)       ND          ND         NA      p.R227Q/p.R227Q
11 (g)       ND          ND         NA      p.R227Q/p.R227Q
12           ND          ND         NA             ND
13         230.5        8.1        28.5            ND
14           ND          ND         NA     c.548-1G>A/p.A228V
15           ND          ND         NA      p.R246Q/p.R246Q
16 (g)       ND          ND         NA      p.G203S/p.G203S

(a) To convert from ng/dL to nmol/L: T, multiply by 0.0347;
DHT, multiply by 0.0344.

(b) Diagnostic cutoff for 5ARD is >17.

(c) ND, not done; NA, not applicable.

(d) Metabolite ratios within age-matched reference intervals.

(e) At 3 days old.

(f) At 5 months old.

(g) Chan et al. (10).

(h) At 17 months old.

(i) At 5 years old.

(j) At 18 days old.

(k) At 2 months old.

Table 2. Genotypic sex-matched reference intervals of
urinary steroid metabolite ratios.

Age group        n    5[alpha]-   11-OH Ad/      Ad/Ae     5[alpha]-
                       THF/THF     11-OH Ae                 THB/THB

3 months
  to 6 years     24   1.01-3.33   2.80-19.73   0.56-2.80   1.88-4.68
7-10 years       31   0.80-4.25   4.82-33.42   0.67-4.09   1.51-3.31
11-1 7 years     44   0.94-2.26   4.05-57.64   1.11-3.49   1.19-3.38
18-40 years      83   0.48-2.53   1.09-38.45   0.67-2.96   0.84-3.50

Table 3. Hormonal and genetic findings in patients with 5ARD reported
in the medical literature between 2003 and 2012.

                                   Patients with
                                     baseline/
                                post-hCG-stimulated
                    Patients,    DHT measurements,
Ethnic group          n (a)              n

Diverse                34              17/13
Diverse                20              13/7
Diverse              14 (c)            ND/ND
Mexican-Mestizo        11              ND/ND
Chinese                 8               0/0
Egyptian                8               6/5
Chinese               6 (c)             0/0
Italian                 6               5/0
Korean                  5               3/3
Cypriot                 5               4/4
Diverse                 4               3/0
Pakistani             4 (c)             2/3
Diverse                 4               3/2
Chinese                 3               1/3
Mexico                  3               1/2
Egyptian                2               2/2
Italian, American       2               2/0
Thai                    2               1/1
Korean                  2               1/1
Turkish                 2               2/2
Turkish                 1               1/0
German                  1               0/1
Spanish                 1               1/1
Chinese                 1               1/0

                     Diagnostic cutoff
                    used for baseline/
                    post-hCG-stimulated       DHT
Ethnic group            T/DHT ratio       methodology

Diverse                  ND (b)/10            RIA
Diverse                     ND                RIA
Diverse                     ND                ND
Mexican-Mestizo             ND                ND
Chinese                     NA                NA
Egyptian                    ND                RIA
Chinese                     NA                NA
Italian                     ND                RIA
Korean                  ND/3.6-7.0            RIA
Cypriot                     ND                ND
Diverse                     ND                ND
Pakistani                   ND                ND
Diverse                     ND                ND
Chinese                  <10.5 (d)            RIA
Mexico                      ND                ND
Egyptian                    ND                ND
Italian, American        10.5/8.5             RIA
Thai                        ND               ELISA
Korean                      ND                RIA
Turkish                     ND                ND
Turkish                   <12/ND              ND
German                    <18 (d)            GCMS
Spanish                     ND                RIA
Chinese                     ND                ND

                    Patients with T/DHT
                      ratio above the       Patients
                    diagnostic cutoff at   genetically
                     baseline/after hCG    confirmed,
Ethnic group           stimulation, n           n

Diverse                    ND/10               33
Diverse                      NA                15
Diverse                      NA                14
Mexican-Mestizo              NA                 9
Chinese                      NA                 8
Egyptian                     NA                 8
Chinese                      NA                 6
Italian                      NA                 6
Korean                      NA/2                5
Cypriot                      NA                 5
Diverse                      NA                 4
Pakistani                    NA                 4
Diverse                      NA                 4
Chinese                     1/3                 3
Mexico                       NA                 3
Egyptian                     NA                 2
Italian, American           2/NA                2
Thai                         NA                 2
Korean                       NA                 2
Turkish                      NA                 2
Turkish                     1/NA                1
German                      NA/0                1
Spanish                      NA                 1
Chinese                      NA                 1

Ethnic group        Reference

Diverse             Maimoun et al. (2)
Diverse             Hackel et al. (11)
Diverse             Berra et al. (12)
Mexican-Mestizo     Vilchis et al. (13)
Chinese             Nie et al. (14)
Egyptian            Mazen et al. (15)
Chinese             Chan et al. (10)
Italian             Baldinotti et al. (3)
Korean              Ko et al. (4)
Cypriot             Skordis et al. (16)
Diverse             Maimoun et al. (17)
Pakistani           Perry et al. (5)
Diverse             Maimoun et al. (6)
Chinese             Zhang et al. (18)
Mexico              Vilchis et al. (19)
Egyptian            Hafez et al. (20)
Italian, American   Bertelloni et al. (21)
Thai                Sahakitrungruang et al. (22)
Korean              Kim et al. (23)
Turkish             Bahceci et al. (24)
Turkish             Savas Erdeve et al. (25)
German              Walter et al. (26)
Spanish             Fernandez-Cancio et al. (27)
Chinese             Fernandez-Cancio et al. (28)

(a) Only those patients first reported in the literature review period.

(b) ND, not documented; NA, not applicable.

(c) Patients in whom USP was performed.

(d) Baseline or post-hCG stimulation not specified.
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Title Annotation:Endocrinology and Metabolism
Author:Chan, Angel On Kei; But, Betty Wai Man; Lee, Ching Yin; Lam, Yuen Yu; Ng, Kwok Leung; Tung, Joanna Y
Publication:Clinical Chemistry
Article Type:Report
Geographic Code:9HONG
Date:May 1, 2013
Words:5439
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