Pao Lin Kuo

The Expression Level of Septin12 Is Critical for Spermiogenesis

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Adrenal myelolipoma with translocation (3;21)(q25;p11)

Adrenal myelolipoma (ML) is a rare, benign, nonfunctioning tumor-like lesion composed of mature adipose tissue interspersed with bone marrow-like hematopoietic elements in various proportions. It occurs usually in adults and is frequently asymptomatic in about half of cases. The histogenesis of adrenal ML is not clear and this lesion has been found to be associated with endocrine disorders, other adrenal dysfunction and tumors, and hyperstimulation with adrenocorticotropic hormone. Specific chromosomal abnormalities, however, have not been observed in such cases. Herein, we report a typical case of adrenal ML found incidentally in a 26-year-old man. Conventional cytogenetic techniques demonstrated balanced translocation between bands 3q25 and 21p11 in 9 of 20 metaphases analyzed in cultured tumor cells. To the best of our knowledge, this is the first reported case of adrenal ML showing chromosomal abnormality. This finding would indicate that adrenal ML is a bona fide neoplasm and the possibility of derivation from misplaced hematopoietic cells may be alternatively taken into consideration in view of the similar genetic changes in hematopolietic neoplasms.

SEPT12 mutations cause male infertility with defective sperm annulus.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis‐specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine‐5′‐triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12D197N) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild‐type SEPT12 in a dose‐dependent manner. The patient carrying SEPT12D197N presented with oligoasthenozoospermia, whereas the SEPT12T89M patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12D197N patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss‐of‐function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation. Hum Mutat 33:710–719, 2012.

Presence of DAZL transcript and protein in mature human spermatozoa

OBJECTIVE To identify the DAZL transcript and protein location in human spermatozoa. DESIGN In vitro experiment. SETTING University-based reproductive genetics laboratory. PATIENT(S) A fertile volunteer. INTERVENTION(S) Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining for DAZL. MAIN OUTCOME MEASURE(S) Expression of DAZL in human spermatozoa. RESULT(S) The DAZL-specific primers yield a 128 bp product in ejaculate. A protein of approximately 33.5 kDa was detected by Western blot analysis. Immunofluorescence staining showed strong homogeneous staining in the midpiece of spermatozoa and weak staining in the principal piece. A speckled-type distribution was found in the head region. CONCLUSION(S) The DAZL transcript and protein are present in human spermatozoa. The roles of DAZL protein in sperm motility and in the sperm-oocyte interaction await further investigation.

Association of polymorphisms/haplotypes of the genes encoding vascular endothelial growth factor and its KDR receptor with recurrent pregnancy loss

BACKGROUND Vascular endothelial growth factor (VEGF) and its kinase insert domain receptor (KDR) play an important role in angiogenesis, and their gene expression patterns also suggest a close relationship with early pregnancy. However, limited information is available regarding the role of the VEGF system, especially its KDR receptor, in recurrent pregnancy loss (RPL). This study was conducted to investigate a genetic association between VEGF and its receptor gene (KDR) with idiopathic RPL. METHODS In this case-control study, 115 women who had experienced at least two consecutive spontaneous miscarriages (n= 62 women with two miscarriages, n= 53 with three or more) and 170 controls were included. A total of 14 tag single-nucleotide polymorphisms (SNPs) of VEGF and KDR were selected from the HapMap Web site and three functional SNPs [rs1570360 (-1154G/A) of VEGF; rs2305948 (V297I) and rs1870377 (Q472H) of the KDR gene] were genotyped using primer extension analysis. We further used multifactor dimensionality reduction analysis to evaluate gene-gene interactions. RESULTS One tag SNP (rs6838752) and the functional SNP (Q472H) of the KDR gene were in complete linkage and showed significant differences between patients and controls (P< 0.05). The frequencies of haplotypes of VEGF (A-T-G haplotype) and KDR (A-C-A-T-G haplotype) showed significant differences in patients versus controls (P< 0.05). All comparisons with controls remained significant when the subgroup of women with three or more miscarriages was analyzed. CONCLUSIONS VEGF and its receptor gene (KDR) are associated with idiopathic RPL. The VEGF/KDR system jointly contributes to recurrent miscarriage in Taiwanese Han women.

Genetic association studies of ACE and PAI-1 genes in women with recurrent pregnancy loss: a systematic review and meta-analysis.

A fine balance between coagulation and fibrinolysis is critical in early pregnancy. Plasminogen activator inhibitor-1 (PAI-1) and angiotensin converting enzyme (ACE) are involved in the fibrinolytic process, and several studies have reported the association between their gene polymorphisms and recurrent pregnancy loss (RPL). This study was conducted to investigate the association between PAI-1 and ACE polymorphisms and idiopathic RPL, using meta-analyses. A systematic review of the published literature from the MEDLINE and EMBASE databases before April 2012 was conducted. Of 209 potentially relevant studies, 22 case-control studies comprising a total of 2,820 RPL patients and 3,009 controls were included. Among these studies were 11 reports of PAI-1 4G/5G and 11 of ACE I/D polymorphisms in patients with RPL. A significant association was found withthe ACE I/D polymorphism [summary odds ratio 1.29 (95% confidence interval 1.02-1.62)] in studies including more than two recurrent abortions. Subgroup analysis did not show significant associations with RPL in Caucasian and non-Caucasian patients. Meta-analyses of PAI-1 4G/5G polymorphism were not found associations with RPL in studies including more than two or three recurrent abortions, and in studies of Caucasian and non-Caucasian patients. In conclusion, meta-analyses showed a significant association between the ACE I/D polymorphism and idiopathic RPL. High clinical heterogeneity existed among studies of PAI-1 4G/5G, and the aggregated data failed to confer higher susceptibility to idiopathic RPL. More well-designed studies with different ethnic populations are required for future integration.

Polymorphisms of endocrine gland-derived vascular endothelial growth factor gene and its receptor genes are associated with recurrent pregnancy loss

BACKGROUND Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its receptor genes [prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2)] have been identified in the last decade and their expression is restricted to the steroidogenic glands (ovary, testis, adrenal gland and placenta). Their expression patterns also suggest a close relationship to early pregnancy. However, little information is available regarding the role of EG-VEGF and its receptors (PKR1 and PKR2) in recurrent pregnancy loss (RPL). This study was conducted to investigate the association between polymorphisms of EG-VEGF and its receptor genes (PKR1 and PKR2) and idiopathic RPL. METHODS In this case-control study, 115 women with a history of idiopathic RPL and 170 controls were included. A total of 11 tag single nucleotide polymorphisms (SNPs) selected from EG-VEGF, PKR1 and PKR2 were genotyped. We further used multifactor dimensionality reduction (MDR) analysis to choose a best model and evaluate gene-gene interactions. RESULTS Two tag SNPs of PKR1 (rs4627609, rs6731838) and one tag SNP of PKR2 (rs6053283) were significantly associated with idiopathic RPL (P < 0.05). The frequencies of haplotypes C-G and T-A of PKR1 and haplotype A-G-C-G-G of PKR2 were significantly increased in women with idiopathic RPL (P < 0.05); MDR tests revealed gene-gene interactions between three loci [EG-VEGF (rs7513898), PKR1(rs6731838), PKR2(rs6053283)] based on the association model (P = 0.008). The adjusted odds ratio of high- and low-risk genotype combinations in the three-locus model was 3.94 (95% confidence interval: 2.38-6.52). CONCLUSIONS EG-VEGF receptor (PKR1, PKR2) gene polymorphisms and haplotypes were associated with idiopathic RPL. These three genes (EG-VEGF, PKR1 and PRK2) jointly contribute to RPL in the Taiwanese Han population.

Polymorphisms of estrogen-related genes jointly confer susceptibility to human spermatogenic defect

OBJECTIVE To establish a multilocus model for studying the effect of estrogen-related genes on impaired spermatogenesis. DESIGN Prospective study. SETTING University-based reproductive clinics and genetics laboratory. PATIENT(S) A total of 183 oligozoospermatic (sperm count <20 x 10(6)/mL) or azoospermatic males and 120 fertile control males were included. INTERVENTION(S) A total of 16 single nucleotide polymorphisms (SNPs) from nine genes (estrogen receptors [ER-alpha, ER-beta], estrogen synthesizing/metabolizing genes [CYP17, CYP19A1, HSD17B2, CYP1A1, CYP1B1, COMT], and transport genes [SHBG]) were genotyped. The combinatorial effect of multiple genetic variants was assessed using the multilocus model. MAIN OUTCOME MEASURE(S) Significantly associated SNPs and odds ratio (OR). RESULT(S) Six SNPs from five genes (rs180113 of ER-alpha gene, rs1256049 of ER-beta gene, rs1048943 of CYP1A1 gene, rs8191246 of HSD17B2 gene, and rs1799941 along with rs6259 of SHBG gene) were found to be significantly associated with spermatogenic defect. The genes were further divided into three categories according to their functions (receptors, synthesis and metabolism, and transporter). Based on our multilocus risk model, men with risk alleles in two of the three gene families had increased risk of impaired sperm production (OR = 10.5). The OR further increased to 34.6 for men with unfavorable alleles for all three gene families. CONCLUSION(S) Polymorphisms of estrogen-related genes jointly confer susceptibility to human spermatogenic defect at the prereceptor, receptor, and postreceptor levels in the Taiwanese Han population.

Quantitative trait analysis suggests polymorphisms of estrogen-related genes regulate human sperm concentrations and motility

BACKGROUND Human spermatogenesis is regulated by complex networks, and estrogens are recognized as one of the significant regulators of spermatogenesis. We tested the associations between variants of estrogen-related genes and semen parameters. METHODS We performed genotyping for genetic variants of estrogen-related genes and quantitative trait analysis of fertile and infertile men with well-characterized reproductive phenotypes. Men with known semen parameters (n= 677) were enrolled, including 210 fertile men and 467 infertile men. A total of 17 genetic markers from 10 genes, including 2 estrogen receptors (ER-α, ER-β), 7 estrogen synthesizing/metabolizing genes (CYP19A1, HSD17B1, CYP1A1, CYP1B1, COMT, GSTM1, GSTT1) and 1 transport gene (SHBG) were genotyped. Sperm concentration, motility and morphology were taken as quantitative traits to correlate with genetic variants in the estrogen-related genes. RESULTS Five genes (rs1801132 and rs2228480 of the ER-α gene, rs1256049 and rs4986938 of the ER-β gene, rs605059 of the HSD17B1 gene, rs1799941 of the SHBG gene and rs1048943 and rs4646903 of the CYP1A1 gene) were found to be significantly associated with sperm concentration (P< 0.01), while five genes (rs1801132 of the ER-a gene, rs1256049 of the ER-β gene, rs1048943 of the CYP1A1 gene, rs605059 of the HSD17B1 gene and rs1799941 along with rs6259 of the SHBG gene) were associated with sperm motility (P< 0.01). None of the estrogen-related genes were associated with sperm morphology. With an increasing number of risk alleles, sperm concentration and motility tended to deteriorate and show a loci-dosage effect. CONCLUSIONS Quantitative trait analysis based on a limited number of genetic markers suggests that estrogen-related genes mainly regulate sperm concentration and motility.

Y-chromosome microdeletion and its effect on reproductive decisions in Taiwanese patients presenting with nonobstructive azoospermia

OBJECTIVES To investigate the position, extent, and frequency of Y chromosome microdeletions in Taiwanese patients presenting with nonobstructive azoospermia, and to investigate the effect of microdeletions on reproductive decisions. METHODS We studied 176 consecutive men with azoospermia in our urology clinic. Polymerase chain reaction tests were performed in 94 patients with nonobstructive azoospermia, and a series of 27 sequence-tagged sites (STSs) mapped within intervals 5 and 6 of Yq11 was selected for analysis. Clinical genetics counseling was provided to couples with microdeletions, and these couples made their own choices about further treatment modalities. RESULTS Among 94 patients screened for microdeletion, 11 (11.7%) showed microdeletions of one or more STSs. One had a deletion confined to the azoospermia factor b (AZFb) region (encompassing the RBM gene). Two were found to have deletions of both the AZFb and AZFc regions. Eight patients had deletions in the AZFc region (encompassing the DAZ gene). Five had deletions distal to the DAZ gene family. One had multiple, noncontiguous deletions. In 8 patients with testicular histology available, a lack of genotype/phenotype correlation was noted. Of the 11 couples with deletions, 3 thought microdeletion was a serious defect and opted for an artificial insemination of donor or adoption, 5 chose intracytoplasmic sperm injection, and the other 3 decided to undergo treatment with Chinese medicinal herbs. CONCLUSIONS The most commonly deleted region in the Taiwanese population is AZFc. The genes implicated in Taiwanese spermatogenesis defects are the DAZ and RBM gene families. Twenty-seven percent of couples with microdeletions deferred assisted reproductive technologies because of concern about their underlying genetic defects.