[Comparative analysis of two assaysin detection of sperm DNA fragmentation index, flow cytometry and AI-based fluorescence microscopy, based on AO staining: A multicentre study].

OBJECTIVE: To study the correlation, consistency, and variations between two assays of DNA fragmentation index based on acridine orange (AO) staining via AI-based fluorescence microscopy(AI-DFI), and flow cytometry (FCM-DFI) across multiple centers. METHODS: We selected 421 male patients from Nanjing Drum Tower hospital ( Hospital G) (226 cases), Eastern Theatre General Hospital (Hospital J) (89 cases) and Jiangsu Province Hospital (Hospital S) (106 cases) . Semen samples from each patient were analyzed for routine semen parameters and for DFI using both AI fluorescence microscopy and flow cytometry. We studied the two methods' stability as well as the correlation, consistency, and variation between the two methods' results in various centers. RESULTS: The two replicate studies' results of AI-DFI and the three centers' FCM-DFI for linear regression analysis indicated strong stability (R2>0.9).Overall(Group A), the AI-DFI results demonstrated good correlation and consistency with the FCM-DFI results of three centers (r>0.85；ICC>0.9).The semen specimens were categorized into two groups: normal specimen group (group B) and abnormal specimen group (group C) (including asthenozoospermia, oligospermia, and semen samples with high impurities).Group C's results showed a decline in correlation and consistency when compared to group A and group B, whereas group B's results showed a little rise in correlation and consistency when compared to group A. Although the consistency and correlation between the results of the two DFI testing methods in the three centers were good, there was still a significant difference between Groups A and C (P<0.05), and in Group B there was a significant difference between the two DFI testing methods only in Hospital G (p＝0.02), with no significant difference in Hospitals J and S (P> 0.05). CONCLUSION: The two detection methods exhibit good stability and correlation. However, significant differences are observed in the DFI detection methods in samples with abnormal semen parameters and high complexity. The main reason for these significant differences may lie in the variations in detection principles. Each detection method has its own advantages, allowing clinical or research settings to choose between them based on laboratory conditions or specific requirements.

[Effects of sperm DNA fragmentation index on semen parameters and pregnancy outcomes in intrauterine insemination].

Objective: To analyze the correlation of the sperm DNA fragmentation index (DFI) level with semen parameters and pregnancy outcomes of artificial insemination of the husband (AIH) in the cycle of intrauterine insemination (IUI). METHODS: We collected the clinical data on 777 cases of IUI, including female clinical indicators, male semen parameters, sperm DFI and pregnancy outcomes. According to the DFI level, we divided the patients into three groups: DFI < 15%, 15% ≤ DFI < 30% and DFI ≥ 30%. RESULTS: The sperm DFI level was significantly elevated with the increased age of the males (P = 0.002) and closely related to the total number of motile sperm (P = 0.002) and total sperm motility (P = 0.000) before treatment, as well as to sperm concentration (P = 0.000), total sperm motility (P = 0.001) and total number of progressively motile sperm (P = 0.000) after density gradient centrifugation. The rate of clinical pregnancy was decreased in the DFI ≥ 30% group. There were no statistically significant differences between sperm DFI and the rates of clinical pregnancy and abortion. CONCLUSIONS: Male age significantly affects the sperm DFI level. Sperm DFI is closely related to sperm motility and total number of progressively motile sperm, but not to the rates of clinical pregnancy and abortion in patients undergoing IUI. IUI can be used as an effective method of assisted reproduction for male infertility./.

[HONO Observation and Assessment of the Effects of Atmospheric Oxidation Capacity in Changzhou During the Springtime of 2017].

HONO measurement was conducted using a wet-chemistry-based method at the Changzhou Environmental Monitoring Center in April 2017. HONO ranged from 0.2-13.9 µg·m-3 with an average of (2.9±2.3) µg·m-3. O3, HCHO, volatile organic compounds, photolysis frequency, and meteorological parameters were simultaneously monitored.·OH concentration was simulated by a Master Chemical Mechanism box model and the daytime maximum·OH concentration ranged from 1.0×106 to 14×106 molecules per cubic centimeter. The formation rates of·OH by photolysis of HONO, O3, HCHO, H2O2, and alkene ozonolysis were calculated as well. The effects of the five sources on atmospheric oxidation capacity were revealed:O3 photolysis (46.4%) > HONO photolysis (41.1%) > alkene ozonolysis (10.9%) > HCHO photolysis (1.5%) > H2O2 photolysis (0.1%). HONO photolysis for OH radical production played a major role in the early morning, before with an increase in O3 concentration, O3 photolysis began to account for most of the·OH production. After 17:00, due to a significant decrease in the intensity of solar radiation, the alkene ozonolysis started playing a major role in the formation of·OH. The photolysis of formaldehyde and hydrogen peroxide played a negligible role in·OH radical production in this study.

Analysis of human sperm DNA fragmentation index (DFI) related factors: a report of 1010 subfertile men in China.

BACKGROUND: Many factors may lead to sperm DNA damage. However, it is little known that the correlations of sperm DNA damage with obesity-associated markers, and reproductive hormones and lipids levels in serum and seminal plasma. METHODS: In our prospective study, a total of 1 010 subfertile men, aged from 18 to 50 years old, were enrolled from August 2012 through June 2015. Their obesity-associated markers, semen parameters, sperm acrosomal enzyme activity, seminal plasma biochemical markers, and reproductive hormones and lipids levels in serum and seminal plasma were detected. Sperm DNA fragmentation index (DFI) was determined by sperm chromatin structure assay. The correlations between DFI and each of the above-mentioned variables were analyzed. RESULTS: Spearman correlation analysis showed that sperm DFI was positively related to age and abstinence time (P<0.001). Sperm DFI was also positively related to semen volume and percent of abnormal sperm head (P<0.001), while negatively related to sperm concentration, progressive motility (PR), sperm motility, total normal-progressively motile sperm count (TNPMS), percent of normal sperm morphology (NSM), percent of intact acrosome and acrosomal enzyme activity (P<0.001). Sperm DFI was positively related to seminal plasma zinc level (P<0.001) but unrelated to seminal plasma total α-glucotase, γ-glutamyl transpeptidase (GGT) and fructose levels. There was no any correlation between sperm DFI and obesity-associated markers such as body mass index (BMI), waist-to-hip ratio (WHR), waist circumference (WC) and waist-to-height ratio (WHtR), and serum lipids levels, but there was positive correlation between sperm DFI and seminal plasma triglyceride (TG) and total cholesterol (TC) levels (P<0.001). Sperm DFI was positively related to serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and seminal plasma FSH and estradiol (E2) levels (P<0.001), but unrelated to serum and seminal plasma testosterone (T) levels. The multivariate regression analysis for the variables which were significantly correlated with sperm DFI in Spearman correlation analysis showed that age, semen volume, sperm concentration, progressive motility, TNPMS and intact acrosome were independently correlated with sperm DFI. CONCLUSIONS: There are many potential factors associated with sperm DFI, including age, abstinence time, spermatogenesis and maturation, seminal plasma lipids and reproductive hormones levels. However, the potential effects of seminal plasma lipids and reproductive hormones on sperm DNA damage need still to be demonstrated by the studies with scientific design and a large size of samples.

Relation of size of seminal vesicles on ultrasound to premature ejaculation.

Myriad biological factors have been proposed to explain premature ejaculation (PE). However, data correlating PE with seminal vesicles (SVs) are sparse. The study aimed to evaluate the relationship between the size of SV and PE. The cross-sectional study included 44 outpatients with PE and 44 volunteers without PE, and the size of SV was compared. Self-estimated intravaginal ejaculatory latency time, the Premature Ejaculation Diagnostic Tool (PEDT), the International Index of Erectile Function-15, and the National Institutes of Health-Chronic Prostatitis Symptom Index were used for assessment of symptoms. Compared to the control group, the PE group had significantly higher mean anterior-posterior diameter (APD) of SV (P < 0.001). The optimal mean APD of SV cutoff level was 9.25 mm for PE. In the PE group, PEDT was also higher with a mean APD of SV ≥9.25 mm compared with mean APD of SV <9.25 mm. PEDT was significantly correlated with the mean APD of SV (r = 0.326, P = 0.031). The seminal plasma proteins were compared between six PE and six matched control cases by mass spectrometry and it was shown that 102 proteins were at least 1.5-fold up- or down-regulated. Among them, GGT1, LAMC1, and APP were significantly higher in the PE group. These results indicated that men with a larger mean APD of SV might have a higher PEDT score. Transrectal ultrasound of SV should be considered in the evaluation of patients with premature ejaculation. SV might be a potential target for the treatment of patients with PE and ultrasound change in SV.

[Effect of secretin on the expression of cPLA2 and mPGEs-1 in mouse endometrial stromal cell during early pregnancy].

Secretin, a gastrointestinal peptide, has been found to be expressed in mouse endometrial stromal cells (mESCs) during early pregnancy. In order to further investigate the function of secretin during embryo implantation, the expression levels of secretin, secretin receptor, cytosolic phospholipase A2 (cPLA2) and membrane prostaglandin E synthase 1 (mPGEs-1) were detected in the mice uterus from day 4 to 8 of pregnancy by real-time PCR, ELISA and in situ hybridization. mESCs isolated and cultured from day 4 of pregnancy were transfected with secretin expression vectors or treated with H89, a PKA inhibitor. Then the expression levels of cPLA2, mPGEs-1 and cAMP responsive element-binding protein (CREB) were detected by real-time PCR and Western blot. The concentration of prostaglandin E2 (PGE2) in the supernatant was determined by ELISA. The result showed that secretin, cPLA2 and mPGEs-1 mRNA expression increased gradually in implantation sites from day 5 to day 7 of pregnancy with the same tendency. The secretin levels in serum were significantly higher on days 6, 7 and 8 of pregnancy than that on day 5 of pregnancy. The concentration of secretin was significantly higher in implantation sites on days 6, 7 than that in non-implantation site on day 5. Transfection of secretin expression vector promoted cPLA2, p-cPLA2 and mPGEs-1 expressions in mESCs, but not PGE2 level in the supernatant. H89 could effectively inhibit the expression of CREB, p-CREB, p-cPLA2 and cPLA2 induced by secretin. The results showed that the increased secretin expression in mESCs during embryo implantation may promote p-cPLA2, cPLA2 and mPGEs-1 expression, and the promotion may be through PKA signaling pathway.

Relationship between Lipids Levels of Serum and Seminal Plasma and Semen Parameters in 631 Chinese Subfertile Men.

OBJECTIVE: This prospective study was designed to investigate the relationship between lipids levels in both serum and seminal plasma and semen parameters. METHODS: 631 subfertile men were enrolled. Their obesity-associated markers were measured, and semen parameters were analyzed. Also, seminal plasma and serum TC, TG, HDL and LDL and serum FFA, FSH, LH, total testosterone (TT), estradiol (E2) and SHBG levels were detected. RESULTS: Seminal plasma and serum TG, TC and LDL levels were positively related to age. Serum TC, TG and LDL were positively related to obesity-associated markers (P < 0.001), while only seminal plasma TG was positively related to them (P < 0.05). For lipids levels in serum and seminal plasma, only TG level had slightly positive correlation between them (r = 0.081, P = 0.042). There was no significant correlation between serum lipids levels and semen parameters. However, seminal plasma TG, TC, LDL and HDL levels were negatively related to one or several semen parameters, including semen volume (SV), sperm concentration (SC), total sperm count (TSC), sperm motility, progressive motility (PR) and total normal-progressively motile sperm counts (TNPMS). Moreover, seminal plasma TG, TC, LDL and HDL levels in patients with oligospermatism, asthenospermia and teratozoospermia were higher than those with normal sperm concentration, motility or morphology. After adjusting age and serum LH, FSH, TT, E2 and SHBG levels, linear regression analysis showed that SV was still significantly correlated with seminal plasma LDL (P = 0.012), both of SC and TSC with seminal plasma HDL (P = 0.028 and 0.002), and both of PR and sperm motility with seminal plasma TC (P = 0.012 and 0.051). CONCLUSION: The abnormal metabolism of lipids in male reproductive system may contribute to male factor infertility.

Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3ß-HSD, and 17ß-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3ß-HSD, and 17ß-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 µmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3ß-HSD, and 17ß-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3ß-HSD, and 17ß-HSD in Leydig cells.

Annexin V-induced rat Leydig cell proliferation involves Ect2 via RhoA/ROCK signaling pathway.

This study investigated the effect of annexin V on the proliferation of primary rat Leydig cells and the potential mechanism. Our results showed that annexin V promoted rat Leydig cell proliferation and cell cycle progression in a dose- and time-dependent manner. Increased level of annexin V also enhanced Ect2 protein expression. However, siRNA knockdown of Ect2 attenuated annexin V-induced proliferation of rat Leydig cells. Taken together, these data suggest that increased level of annexin V induced rat Leydig cell proliferation and cell cycle progression via Ect2. Since RhoA activity was increased following Ect2 activation, we further investigated whether Ect2 was involved in annexin V-induced proliferation via the RhoA/ROCK pathway, and the results showed that annexin V increased RhoA activity too, and this effect was abolished by the knockdown of Ect2. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated annexin V-induced proliferation and cell cycle progression. We thus conclude that Ect2 is involved in annexin V-induced rat Leydig cell proliferation through the RhoA/ROCK pathway.

[Protective effect of epigallocatechin gallate against sperm abnormality in mice].

OBJECTIVE: To investigate the protective effect of epigallocatechin gallate (EGCG) on mouse sperm in vivo. METHODS: A total of 64 six-week-old male Kuming mice were randomly divided into eight groups of equal number to be treated with normal saline (negative control), Cyclophosphamide (CP) at 30 mg/kg (positive control), and CP followed by EGCG (experimental) at 20, 40, and 80 mg/kg, respectively, given every other day for 10 days. At 4 and 5 weeks after treatment, the bilateral testes of the mice were harvested for examination of sperm abnormality. RESULTS: EGCG did not increase the rate of CP-induced sperm abnormality in the mice, but reduced it instead with the prolonged time of treatment. CONCLUSION: EGCG protects mouse sperm in vivo.

[Waist-hip ratio correlates with the levels of reproductive endocrine hormones in abdominally obese males].

OBJECTIVE: To study the impact of abdominal obesity on the production of male reproductive endocrine hormones. METHODS: This study included 342 male patients at the andrology clinic, aged 19 -47 years and higher than 160 cm. We measured their waistlines, hiplines and waist-hip ratio, detected the levels of serum estradiol (E2), testosterone (T), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and free testosterone (FT) by chemiluminescence and radioimmunoassay, and analyzed the correlation of the waist-hip ratio with the levels of reproductive endocrine hormones. Abdominal obesity was defined as the waist-hip ratio > 0.9. RESULTS: In the 342 male patients, there were 62 cases of abdominal obesity and 280 cases of the normal somatotype (waist-hip ratio < or = 0.9). The waist-hip ratio was negatively correlated with the T level (r = -0.163, P = 0.003) and the T/LH ratio (r = -0.13, P = 0.02). Both the T level and T/LH ratio were significantly reduced in the abdominal obesity patients ([14.51 +/- 4.53] nmol/L and 2.26 +/- 0.36) as compared with the normal somatotype controls ([17.21 +/- 4.23] nmol/L and 4.61 +/- 0.19) (P < 0.05). CONCLUSION: The waist-hip ratio has a significant negative correlation with the T level and T/LH ratio, and the serum T level is significantly lower in men with abdominal obesity than in those of the normal somatotype.

[Effect of annexin 5 on StAR and testosterone synthesis related enzyme in rat Leydig cells].

OBJECTIVE: To investigate the effect of annexin 5 on the expressions at mRNA levels and protein levels of StAR, P450scc, 3ß-HSD, 17α-hydroxylase and 17ß-HSD in rat Leydig cells. METHODS: The primary rat Leydig cells were cultured for 24 h and then stimulated with 10(-9) mol/L annexin 5 for 12 h and 24 h respectively. Cellular total RNA and total protein were extracted respectively. The expressions of StAR, P450scc, 3ß-HSD, and 17α-hydroxylase and 17ß-HSD(10) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR)and the protein levels were detected by Western blotting. RESULTS: Compared with the control group, at the mRNA level, after being treated with annexin 5 for 12 h, only 17ß-HSD(10) expression had a 26% increase (P<0.05) while the others had no significant difference. The expressions of StAR, P450scc and 3ß-HSD elevated 55%, 69% and 59%(P<0.05) respectively, and 17ß-HSD(10) increased 104%(P<0.01) while 17α-hydroxylase had no significant difference after being treated with annexin 5 for 24 h. At the protein level, after being treated with annexin 5 for 12 h, 17ß-HSD expression had a 39% increase (P<0.05). After 24 h, P450scc, 3ß-HSD and 17ß-HSD elevated 35%, 88% (P<0.05) and 47% (P<0.01) respectively while StAR had no significant difference. CONCLUSION: Annexin 5 regulates testosterone synthesis by affecting the expressions of P450scc, 3ß-HSD and 17ß-HSD at gene and protein levels.

Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells.

Gonadotropin-releasing hormone (GnRH) is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase (MAPK) pathways are involved in GnRH agonist (GnRHa)-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay (RIA). The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3ß-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L⁻¹ after 24 h, activation of ERK1/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERK1/2, the upregulation of 3ß-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3ß-HSD gene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.

Meiotic pairing error in an infertile male bearing reciprocal deletion of chromosome 13.

A series of complex processes takes place during the first meiotic division, including pairing, synapsis, recombination, and segregation of homologous chromosomes. When any of these processes is altered, cellular checkpoints arrest the progression of meiosis and induce cell loss, causing a severe reduction in fertility, or even sterility. In this study, we report on a 29-year-old, healthy, but severe oligozoospermic male with a supernumerary, ring-neocentric 13q12.3 → 13q22 chromosome and a reciprocal deletion, which interfere with the meiotic pairing of chromosomes 13, causing spermatogenesis failure.

[Mutation analysis of NF2 gene and clinical investigation in a Chinese family with neurofibromatosis type II].

OBJECTIVE: To report a heterozygous RNA-splicing mutation (IVS3+ 3A to C) of NF2 gene in a Chinese family with autosomal dominant neurofibromatosis type II and investigate the relationship between the genotype and phenotype. METHODS: The proband with bilateral vestibular schwannomas underwent gamma knife radiosurgery two years earlier. DNA of blood samples from all affected individuals, suspected individuals and unaffected relatives of the family was extracted and amplified to detect the polymorphisms at loci D22S1150 and D22S268 that are linked with the NF2 gene. Two-point LOD score was calculated. The promoter region, 17 exons and exon/intron boundaries of NF2 gene were amplified and sequenced for the proband. The exon 3/intron 3 boundaries of NF2 gene was amplified and sequenced for the other 3 patients, 1 suspected individual, 9 unaffected members of the family and 150 unrelated controls. RESULTS: The result of two-point linkage analysis suggested that NF2 gene was a candidate gene (Zmax= 2.109, θ = 0.00, locus D22S1150). DNA sequencing revealed a heterozygous splicing mutation in intron 3 (IVS3+ 3A to C) for the proband. Identical mutation was also observed in the other 3 patients and 1 suspected individual. No mutation was found in the 9 normal family members and 150 unrelated controls, which was consistent with the clinical diagnosis. CONCLUSION: This is the first report of familial neurofibromatosis type II with a splicing mutation of IVS3+ 3A to C of the NF2 gene. The mutation might be responsible for the neurofibromatosis type II in the family.

[Effects of semen analysis on human sperm movement parameters at different times after semen collection].

OBJECTIVE: To investigate the effects of the computer-assisted semen analysis (CASA) on human sperm movement parameters at different times after semen collection. METHODS: Ninety-two semen samples with sperm density > or = 20 x 10(6)/ml and sperm liquefaction time < 20 min were placed in a incubation box at the temperature of 37 degrees C. Then the seminal parameters were analyzed with the computer-assisted semen analysis (CASA) system at 20, 30, 60 and 90 min after semen collection. RESULTS: The percentages of grade a and b sperm were significantly lower at 30, 60 and 90 min than at 20 min (P < 0.05), so were the percentages of grade c sperm at 60 and 90 min than at 20 and 30 min (P < 0.05), but there were no significant differences in the percentage of grade c sperm between the 20-min and 30-min groups (P > 0.05). The percentages of grade a + b and a + b + c sperm were also significantly lower at 30, 60 and 90 min than at 20 min (P < 0.05). The beat cross frequency (BCF) was significantly higher at 30 min than at 20 min (P < 0.05), while the lateral head amplitude (ALH) significantly lower at 90 min than at 30 min (P < 0.05). The sperm wobbliness (WOB) was significantly higher while the curvilinear velocity (VCL) significantly lower at 90 min than at 20 and 30 min (P < 0.05). Straightness (STR) at 30, 60 and 90 min, and average path velocity (VAP) and straight line velocity (VSL) at 90 min were significantly lower than at 20 min (P < 0.05). There were no significant differences in sperm density, average motion degree (MAD) and linearity (LIN) among the four groups (P > 0.05). CONCLUSION: The interval between semen collection and sperm routine analysis needs to be standardized. The results of this study suggest that sperm movement parameters of normal liquefied semen samples are relatively constant at 30 -60 min after semen collection.

A case of agonadism associated with y-chromosome rearrangement: cytogenetic and molecular studies.

Testicular regression syndrome (MIM273250) is characterized primarily by absence of gonads in a person of XY karyotype. Phenotypes range from complete female external genitalia (primary or "true" agonadism) to male phenotype with anorchia (testicular regression). Phenotypic differences depend on the stage of embryo development during which testes degenerate. No conclusive mapping can be concluded for the phenotype. We describe a novel case of primary agonadism with a karyotype of 46,X,der(Y)(pter-->q11.23::pter-->p11.31 or p11.2:). Transcriptional analysis revealed little expression of USP9Y and UTY genes on the Y chromosome in our case, which would explain her phenotypes of agonadism with short stature.