hCG Test Research Articles

CHARGE syndrome as unusual cause of hypogonadism: endocrine and molecular evaluation

Coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies (CHARGE) syndrome is a genetic syndrome in which hypogonadism is a frequent feature. A causative mutation within the chromodomain helicase DNA-binding protein-7 gene, which plays an important role in the embryonic development, is present in 2/3 of affected patients. We describe the clinical, hormonal and molecular characteristics of a young man from Ecuador who was diagnosed as having CHARGE syndrome at an adult age. The patient showed several phenotypic features of the syndrome, associated with a prepubertal state and cryptorchidism; hypogonadotrophic hypogonadism with undetectable testosterone levels not responsive to hCG testing and severe osteoporosis were ascertained. Molecular evaluation of the CHD7 gene showed the novel frameshift truncating heterozygous mutation p.Tyr1046Glyfs*23 in exon 12. Magnetic resonance imaging revealed mild hypoplasia of the pituitary gland and hypoplasia of the posterior cranial fossa. Parenteral testosterone therapy led to sexual development over time and, in combination with diphophonate therapy and calcium-vitamin D supplementation, significantly improved bone mineralisation. Early proper hormonal treatment of hypogonadism in patients with complex genetic syndromes is important to achieve normal sexual maturation, improve quality of life and avoid significant comorbidities, such as osteoporosis.

Evaluation of a urinary test as a diagnostic tool of a nonprogressive pregnancy

To evaluate the performance of a one-step test detecting intact hCG and free β-hCG isoforms in the urine of pregnant women to diagnose an abnormal pregnancy. Prospective study. Emergency gynecology departments in teaching hospitals. Five hundred twenty-six patients were enrolled, 272 who were not pregnant and 254 who were pregnant. Semiquantitative determination of intact urinary hCG of supposedly not pregnant and pregnant women with vaginal bleeding and/or vaginal pain between 5 and 8 weeks of amenorrhea. The sensitivity and specificity of the urine test for diagnosing nonpregnancy were, respectively, 100% (252/252) and 100% (272/272). The sensitivity and specificity of the urine test for diagnosing ectopic pregnancy (EP) were, respectively, 97% (32/33) and 83% (142/171). The negative predictive value is 99.3% (142/143). The sensitivity and specificity or the urine test for diagnosing miscarriage were, respectively, 89.6% (43/48) and 83% (142/171). The negative predictive value is 96.6% (142/147). Abnormal pregnancy, such as an EP or a miscarriage, can be rapidly detected with the one-step test for intact hCG and free β-hCG isoforms. If ultrasound cannot confirm the localization and/or evolution of a pregnancy, using this test reduces medical supervision and repeated quantification of hCG.

Gelofusine ® affects the quality control performance of QuickVue ® point-of-care human chorionic gonadotropin test devices

Objective To characterize the influence of Gelofusine ® (succinylated gelatin) on the performance of point-of-care testing (POCT) for human chorionic gonadotropin (hCG) devices. Design and methods Three brands of urine and urine/serum hCG POCT devices were verified. Results Succinylated gelatin affected the performance of the control band in the QuickVue ® hCG POCT devices. Alternative devices were not affected. The hCG test performance is not influenced. Conclusions Gelofusine ® affects specifically the quality control performance of the QuickVue ® hCG POCT devices.

Study the effect of age on HCG test in pregnant women

This study comes to determine the effect of age gender on HCG levels in pregnant women. Fifty-four pregnant women divided into two groups according to age, group1 age range (15 – 30) years, group 2 (31 – 45) years. Pregnant test (β – HCG) applied upon urine sample in laboratory of AL-Sadre educational hospital inAL-NajafCity. The result revealed a highly significant effect (P < 0.05) of age in this hormone.

The concordance between serum anti‐Mullerian hormone and testosterone concentrations depends on duration of hCG stimulation in boys undergoing investigation of gonadal function

In boys undergoing investigation of gonadal function, the relationship between a single measurement of serum anti-Mullerian hormone (AMH) and hCG stimulated serum testosterone is unclear. The aim of the study was to assess concordance between serum AMH and testosterone concentrations following hCG stimulation of two different durations. Samples from 284 children (M : F, 154 : 130) with a median age of 8 years (10th, 90th centiles, 0.25, 14) were used to establish an AMH reference range. Clinical data were reviewed in boys undergoing investigation of gonadal function and who had an AMH measurement and a hCG stimulated (3-day or 3-week) (n = 26) testosterone. Of these 26 boys, 11 had combined genital anomalies, whereas the rest had conditions such as isolated hypospadias, undescended testes or microphallus. Normal testosterone response to hCG stimulation was defined as a level greater than 3.5 nmol at day 4 and 9.5 nmol/l at day 22. In the reference group, the 5th centile AMH for boys below 1 year was 215 pmol/l and between 1 and 8 years 180 pmol/l. The 95th centile for girls for these respective age groups was 30 pmol/l and 25 pmol/l. In those cases where serum testosterone concentrations were available at day 1, day 4 and day 22 of the 3 week-hCG test, five cases had a normal serum testosterone at day 4 and three cases only showed such a response by day 22. In those where serum AMH was less than 180 pmol/l, a poor testosterone response of less than 3.5 nmol was observed in approximately seven of eight (88%) cases with a 3-day hCG stimulation test or the 3-week test. An AMH of greater than 180 pmol/l was associated with a normal testosterone response at day 4 in 10 out of 15 (67%) cases and at day 22 in eight of 11 (73%) cases. However, a low serum testosterone concentration of less than 3.5 nmol after the 3-day hCG test was only associated with a likelihood of a low AMH in three of eight (37%) cases. With the 3-week hCG test, a low day 22 testosterone of 9.5 mmol/l or less was associated with a low AMH of 180 pmol/l or less in four of seven (57%) cases. In boys undergoing investigation of gonadal function, the concordance between AMH and testosterone is better at day 22 than day 4. A normal AMH may provide useful information on overall testicular function but does not exclude the need for an hCG stimulation test.

Gonadal Pathology and Tumor Risk in Relation to Clinical Characteristics in Patients with 45,X/46,Xy Mosaicism

Purpose The decision to perform gonadectomy in patients with 45,X/46,XY mosaicism is often difficult: the increased gonadal tumor risk has to be balanced against the functional capacity of the gonad and the psychological burden of gonadectomy. Material and Methods We combined detailed pathological investigations with corresponding clinical data of 26 45,X/46,XY individuals (43 gonadal specimen), divided into 3 groups according to the external masculinisation score (EMS). Differentiation of gonadal tissue was studied by morphology, and staining for AMH, FOXL2 and SOX9. Tumor risk was assessed based on the differentiation of the gonad, and the presence of malignant or premalignant (immunohistochemical staining for OCT3/4 and/or stem cell factor (SCF)) changes. In males with at least one gonad in situ, results of an HCG test or data on pubertal progression were recorded. Results Testis tubules are SOX9 positive and FOXL2 negative. In undifferentiated gonadal tissue (UGT), co-presence of SOX9 (Sertoli cell differentiation) or FOXL2 (granulosa cell differentiation) is common, with a predominance of FOXL2. No SOX9 or FOXL2 is found in streak gonads. In individuals with atypical genitalia and EMS≥7, indices for (pre)malignancy were found in 1/13 gonads (7.6%), whereas in individuals with atypical genitalia and EMS Conclusions The morphology of UGT is associated with the co-presence of SOX9 and FOXL2 positive cells, normally leading to testicular or ovarian differentiation respectively. In 45,X/46,XY individuals, the clinical phenotype, as evaluated by the EMS, reflects the degree of gonadal “testicularisation” and

Isolated micropenis reveals partial androgen insensitivity syndrome confirmed by molecular analysis

Partial androgen insensitivity syndrome (PAIS) is the milder variant of androgen receptor (AR) defects. The subtle effects of AR mutations present in a patient with micropenis, peno-scrotal hypospadias, infertility, clitoromegaly and posterior labial fusion. We studied the association of isolated micropenis with the genetic defects resulting in androgen resistance, that is, AR gene defects and 5-alpha reductase type 2 (SRD5A2) deficiency. We describe two cases of isolated micropenis: one in a 14-year-old boy and the other in a 3-year-old boy who was followed until he was 10 years old. There were no findings of hypospadias, cryptorchidism or gynecomastia in either of these patients. Serum gonadotrophin and androgen levels were obtained and karyotyping was done. Human chorionic gonadotropin (hCG) stimulation testing assessed the functional capacity of the testes. DNA was extracted from peripheral leukocytes, and all exons of the SRD5A2 and AR genes were amplified by polymerase chain reaction and sequenced. In both patients, baseline testosterone (T) level was low and the values were elevated after hCG testing. The sequence of the SRD5A2 gene was normal in patient 1, and a heterozygous polymorphism, V89L, was found in patient 2. Two known mutations, P390S and A870V, were identified in patients 1 and 2, respectively. Mutations in the AR gene can be associated with isolated micropenis without other features of PAIS, such as hypospadias or gynecomastia. This underlines the importance of including AR gene analysis in the evaluation of isolated micropenis with normal plasma T to ensure proper management of the patient and appropriate genetic counseling for the family.

Testicular choriocarcinoma metastatic to skin and multiple organs. Two case reports and review of literature

Two cases of males with subcutaneous masses as the first clinical sign of testicular choriocarcinoma are reported. The urine and serum human chorionic gonadotropin (hCG) test confirmed high level hCG from male choriocarcinoma. These cases, although very rare, can be a great diagnostic and therapeutic challenge and should be considered in the differential diagnosis of subcutaneous masses occurring in young males.

Two Cases of Bovine Male Pseudohermaphrodites with Different Endocrinological and Pathological Findings

Two cases of bovine male pseudohermaphrodites (PH) were subjected for clinical investigation with transrectal ultrasonography, endocrinology with adoption of hCG-stimulation test, cytogenetics with analysis of sex chromosome and Y-specific DNA, and finally histological examination. Results were compared with normal calves. Case 1 was a 10-month-old calf with XX/XY chimeras, showing elevation of testosterone (T) levels, but no change in progesterone (P(4)) after hCG test, and possessed atrophied testes in the cavitas pelvis. Case 2 was an 18-month-old calf with SRY positive-XY chromosome, showing lower level of plasma T and P(4) after hCG test, and possessed atrophied testes and undifferentiated genital ducts. Both cases possessed female-like external genitalia with similar pathological findings, however endocrinological and cytogenetical aspects were different each other.

Hyaluronan-enriched transfer medium in cleavage-stage frozen-thawed embryo transfers increases implantation rate without improvement of delivery rate

To investigate the efficacy of hyaluronan-enriched transfer media in cleavage-stage frozen embryo transfer cycles. Two commercially available transfer media were prospectively compared in an observational study. Hospital-based in vitro fertilization clinic. Patients (n = 425) undergoing frozen-thawed embryo transfer (FET). The embryos transferred were included in either a study group (high hyaluronic acid [HA], n = 199) or a control group (low HA, n = 226). Delivery rate per FET; positive hCG rate, biochemical pregnancy rate, clinical pregnancy rate, implantation rate, and clinical abortion rate were secondary outcomes. The use of HA in the transfer media significantly increased the positive hCG rate (37.2% vs. 25.2%) and implantation rate (23.1% vs. 15.8%) without increasing the delivery rate (21.6% vs. 21.2%). More subjects in the study group with a positive hCG test experienced biochemical pregnancy (28.4% vs. 8.9%). Addition of HA to transfer media seems to favor attachment of early embryos in FETs without increasing the delivery rate.

How soon can I be proficient in embryo transfer? Lessons from the cumulative summation test for learning curve (LC-CUSUM)

Embryo transfer, a crucial step for achieving pregnancy after in vitro fertilization, is an operator-dependent technique but the number of procedures required for a trainee to reach proficiency is unknown. We set out to evaluate the learning curve (LC) of embryo transfer using a specifically designed statistical tool. The first embryo transfers performed by five trainees were monitored by the cumulative summation test for learning curve (LC-CUSUM), a statistical tool designed to indicate when a process has reached a predefined level of performance. The main outcome measure was a positive hCG test. A 40% pregnancy rate (PR) per transfer was chosen to define adequate performance and a PR of 20% was considered inadequate. After the learning phase, standard CUSUM were implemented to ensure that performance was maintained. The same CUSUM parameters were also applied to monitor 241 consecutive embryo transfers performed by a senior gynaecologist. Between 11 and 99 embryo transfers were necessary for the trainees to reach the predefined level of performance. Simple and intuitive graphical representations of the LCs were generated. CUSUM tests confirmed that performance was maintained after the learning phase. The PR of the senior gynaecologist was 42.7% and the CUSUM showed that performance remained adequate throughout the 241 procedures. This study provides an exportable model for a quantitative monitoring of the LC of embryo transfer as well as a reference curve for continuous monitoring of performance in embryo transfer. The length of the LC of embryo transfer is highly variable, justifying a tailored training to learn this procedure.

Human chorionic gonadotropin tests

Human chorionic gonadotropin (hCG) is not a single biological molecule. There is the regular form of hCG produced by differentiated syncytotrophoblast cells (regular hCG). This hormone primarily functions to maintain the myometrial and decidual spiral arteries, or the vascular supply of the placenta during the full course of pregnancy. Hyperglycosylated hCG is made by undifferentiated cytotrophoblast cells, which are extravillous cytotrophoblast cells. This is an autocrine with separate functions, it maintains trophoblast invasion as in implantation of pregnancy and malignancy in gestational trophoblastic diseases. A hyperglycosylated free β-subunit is produced by a high proportion of all malignancies. This also functions as an autocrine by promoting the growth and invasion of the malignancy. When ordering an hCG test it is important to realize what is being measured and whether the test ordered will detect appropriately these three variants of hCG as well as their degradation products. Most automated commercial laboratory tests, point-of-care tests and over-the-counter tests are limited in what they detect, focusing only on regular hCG. This is in part due to the US FDA, who only consider regular hCG as part of a pregnancy test, and to whom only detection of regular hCG is necessary. This may cause test errors since primarily hyperglycosylated hCG is produced in early pregnancy and in choriocarcinoma and germ cell testicular malignancies. Only free β-subunits may be produced in other germ cell malignancies (all applications for hCG test). The exceptions are the older style hCGb radioimmunoassay and the Siemens Immulite platform hCG test, which detect all β-subunit variants of hCG and their degradation product appropriately. Apart from test specificity limitations, assays for hCG and its variants are widely used clinically in pregnancy detection, early pregnancy detection, prediction of spontaneously aborting, and ectopic pregnancies and prediction of trisomy pregnancies. hCG tests are essential in managing gestational trophoblastic diseases, whether hydatidiform mole, invasive mole or choriocarcinoma, and are very useful in management of testicular malignancies and other germ cell malignancies.

False-positive urine pregnancy test in a woman with adenomysosis

We present a case of a 39-year-old woman presenting to the emergency department with persistent vaginal bleeding with myoma and endometrium thickness. The qualitative urine human chorionic gonadotropin (hCG) showed positive result, however, the quantitative serum hCG had negative result. The negative serum hCG result suggests that the false-positive result was not caused by elevated circulating hCG. According to the urine hCG one-step pregnancy device, 1 mg/dL of hemoglobin may not interference the pregnancy result. Nevertheless, we found that the hemoglobin level was 40 mg/dL in the urine specimen. We designed a in vitro experiment to evaluate the effects of hemoglobin on urine pregnancy test. The concentrations of hemoglobin from 5 to 500 mg/dL did not yield a positive urine pregnancy test. On the basis of our findings, the false positive pregnancy test was not caused by hemoglobin. It is important to confirm a suspected false-positive urine hCG test using a quantitative serum hCG test. Although it is not certain the mechanism for false positive reaction in this peculiar sample, the ACON urine hCG one-step pregnancy will occasionally yield a false-positive result in this class of patients.

Peripheral insulin-like factor 3 concentrations are reduced in men with type 2 diabetes mellitus: effect of glycemic control and visceral adiposity on Leydig cell function

Hypogonadism frequently occurs in men with type 2 diabetes mellitus (T2DM), while the role of glycemic control and visceral obesity is still unclear. This study aimed to assess the Leydig cell function, including the new sensitive marker insulin-like factor 3 (INSL3), in T2DM patients without overt hypogonadism and the influence of either glycemic control or visceral adiposity. Thirty T2DM patients (age 57.1+/-6.2 years, body mass index (BMI) 28.0+/-4.3) without overt hypogonadism and 30 age- and BMI-matched controls were studied. Anthropometric, glycometabolic parameters and testosterone, SHBG, LH, INSL3 levels, bioavailable and free testosterone (BT and cFT) were evaluated. The human chorionic gonadotrophin (hCG) test was also performed. Patients had lower total testosterone (452.6+/-130.0 vs 512.6+/-117.3 ng/dl, P=0.06), BT (189.7+/-36.4 vs 237.1+/-94.1 ng/dl, P=0.002), cFT (8.1+/-1.6 vs 10.1+/-4.0 ng/dl, P=0.002), and higher LH levels (3.5+/-1.6 vs 2.6+/-1.2 mU/ml, P=0.01) versus controls. Serum INSL3 concentrations were also lower in patients (1.1+/-0.3 vs 1.5+/-0.7 ng/ml, P=0.01). These hormonal parameters, including INSL3, did not differ between T2DM patients with poor or good glycemic control (HbA1c>9 or <7% respectively). In patients, waist circumferences (97.9+/-12.4 cm) negatively correlated with INSL3 (P=0.03) and basal, as well as hCG-stimulated testosterone levels (P=0.04 and 0.004 respectively). Basal or stimulated hormonal levels and INSL3 concentrations were not different between patients with (40%) or without erectile dysfunction. An early impairment of the overall Leydig cell function is present in men with T2DM, mainly related to visceral adiposity rather than to glycemic control.

Hyper Sensitive Strip Test with Chemi-luminescence Signal Band

A simple and sensitive method using lateral flow immunoassay (LF) using chemiluminescence reaction has been developed for the detection of antigen. In this work, horseradish peroxidase (HRP) samples were used to confirm the validity of this method and human chorionic gonadotropin (hCG) samples were used to evaluate the limit of detection (LOD) of this method. Through HRP test, this method was confirmed to be reliable for immunoassay, and the results of hCG test show that this method was very sensitive compared with conventional strip test using color of gold nanoparticles or latex beads.

Serum Human Chorionic Gonadotropin Concentrations Greater than 400,000 IU/L Are Invariably Associated with Suppressed Serum Thyrotropin Concentrations

During pregnancy, when human chorionic gonadotropin (hCG) concentrations are highest, there is a transient suppression of serum thyrotropin (TSH). In normal pregnancy, TSH concentrations generally remain within nonpregnant reference intervals; however, in some patients TSH is suppressed. Here we sought to extend previous studies to examine the relationship between very high serum concentrations of hCG (>200,000 IU/L) and the thyroid hormones TSH and free thyroxine (FT(4)). The objective of this study was to determine: 1) if there is an hCG concentration above which TSH concentrations are suppressed (< or =0.2 microIU/mL); 2) how thyroid hormone concentrations change in response to changes in hCG concentrations; and 3) the clinical symptoms in patients with such extremely elevated hCG concentrations. Residual specimens sent to the laboratories for physician-ordered hCG testing were utilized. Over 26 months, 15,597 physician-ordered hCG tests were performed. Sixty-nine specimens from 63 women with hCG concentrations >200,000 IU/L were identified, and TSH and FT(4) concentrations were measured. Medical records were reviewed for clinical information. Thirty-seven percent of subjects had hyperemesis gravidarum (HG) and 19% had gestational trophoblastic disease (GTD). TSH was suppressed (< or =0.2 microIU/mL) in 67% of the specimens with hCG concentrations >200,000 IU/L and 100% of specimens with hCG concentrations >400,000 IU/L. FT(4) concentrations were elevated above the reference interval (1.8 ng/dL) in 32% of specimens with hCG concentrations >200,000 IU/L and in 80% of specimens with hCG concentrations >400,000 IU/L. Only four subjects had documented signs of hyperthyroidism. Women with GTD had a median hCG concentration twofold higher than women with HG and a median TSH concentration one half that of women with HG. 1) At hCG concentrations >400,000 IU/L, TSH is consistently suppressed; 2) serum FT(4) and TSH respond to changes in serum hCG concentrations; and 3) most patients with hCG concentrations >200,000 IU/L lack overt hyperthyroid symptoms.

Micropenis and apparent micropenis--a diagnostic and therapeutic challenge.

Micropenis should be diagnosed shortly after birth and differentiated from associated deformities and syndromes. Calling in a paediatric endocrinologist and a geneticist is obligatory. Endocrine treatment should be undertaken in the second and third months (25 mg testosterone enanthate per month) after a positive HCG test. Where there is a negative endocrine response and/or associated sex differentiation derangement, a sex change operation towards female should be discussed with the family at an early stage. Where male determination is already established and the androgen response is poor or absent, the indication is for plastic surgery to elongate the phallus using the Hinman or Johnston technique, performed by an experienced paediatric urologist.

Effect of an antiestrogen on the testicular response to acute and chronic administration of hCG in normal and hypogonadotropic hypogonadic men: Tamoxifen and testicular response to hCG*

The effect of the antiestrogen tamoxifen (Tx) on the acute and chronic hCG administration was evaluated in patients with hypogonadotropic hypogonadism (HH) and in normal men. An hCG test (5000 IU hCG) was performed before, after two months of hCG administration (2000 IU hCG three times weekly) and after two months of hCG + Tx (2000 IU hCG three times weekly plus 20 mg/day of tamoxifen). Blood samples were obtained before and following 24 and 72 h of every test to determine T, E, 17OHP and SHBG. T increased only in HH with both treatments (X +/- SEM: Basal: 97.9 +/- 19.7; hCG: 237.7 +/- 43.2; hCG +/- Tx: 204.7 +/- 10.7 ng/100 ml). 17OHP rose with hCG alone, but not with hCG + Tx in both groups. E, SHBG and 17OHP/T ratio did not change after treatments. hCG tests: E increased 24 h following hCG administration in every test. The ratio 17OHP/T rose at 24 h in the first and second test but in the third test it did not change. These results support the role of E in the acute hCG-induced Leydig cell desensitization. However, the association of Tx does not improve T serum levels, suggesting that E might not be the unique factor involved in the mechanisms for testicular desensitization.

Hypogonadism of male prolactinomas: Relation to pulsatile secretion of LH: Hypogonadisms des Mannes mit Prolaktinomen: Beziehungen zur pulsatilen LH-Sekretion

In order to investigate whether a hypothalamic disorder cause hypogonadism in male prolactinomas, LH pulsatile secretion was studied in 13 male patients. Serum PRL levels ranged from 186 to 45,000 ng ml-1 before treatment, and all the tumors were macroadenomas. Reduced LH secretion was revealed in 5 of 13 patients, and FSH was reduced in 1 of 13. Serum testosterone (T) levels were lower than the normal limit in all the patients. HCG tests in 3 patients showed good responses, but the peak values of T were lower than those of normal men. LH pulsatilities were examined in 5 hyperprolactinemic patients before treatment, in 4 hyperprolactinemic patients after operation, and in 8 normoprolactinemic patients after operation and/or bromocriptine treatment. There was no significant difference of the mean LH values, the frequencies of LH pulses, and amplitudes among the hyperprolactinemic patients before operation (n = 5), the normoprolactinemic patients after operation (n = 8), and normal men (n = 7). From these results, it was evident that the hypothalamus and pituitary function of male prolactinomas were well preserved, in spite of higher serum PRL levels and larger tumor size than those reported in females. It is suggested that the main cause of hypogonadism in these patients is due to testicular dysfunction resulting from excessive serum PRL.

Plasma concentration of luteinizing hormone, testosterone, dehydroepiandrosterone, androstenedione between birth and one year in the male dog: longitudinal study and hCG stimulation.

A longitudinal study from peripheral blood, with samples collected every week, was performed between birth and one year of age on young Fox terriers dogs in order to determine the patterns of plasma LH, T, DHA and A concentrations. T, DHA, A curves show the same profile. The dog model shows the 3 successive steps preceding the adult life that are met in one year time: First, an infantile period between birth and the 12th week of age: the basal level of LH (4.29 ng/ml) and the 3 androgens levels (T less than 0.3 ng/ml, DHA less than 0.45 ng/ml, A less than 0.36 ng/ml) are low. A pubertal period, between the 13th week and the 36th week of age: we observe the maximum activity of the pituitary gland. The basal level of LH (7.97 ng/ml) significantly increase (P less than 0.001). The mean levels of androgens from 17 to 27 weeks of age are still quite low although significantly higher (P less than 0.03) for T and DHA than previously. After the 27th week of age, the androgens concentrations drastically increase. A post pubertal period begins at the 36th week of age. The mean LH (5.85 nh/ml) decrease. The androgens concentrations seem to plateau during the 12th month of age in the range of 2.5-5 ng/ml for T, 1.5-2.5 ng/ml for DHA and 1-2 ng/ml for A. hCG test (35 UI/kg, IM), with samples collected at 6, 12, 24, 30, 36 and 48 hours post injection, were performed at 1, 4, 7, 9 and 12 months of age. At 1 month, only minor variations were noticed; but after 4 months of age, for the 3 steroids, the same time course response was observed as in the adult dog: maximum levels were reached earlier for DHA (6-12 h) than for T and A (24 h).