Chao Chin Hsu

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.

Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure

OBJECTIVE To develop a simple and rapid protocol for detecting deletions of the Y chromosome and to evaluate the feasibility of gene-based screening in men with spermatogenic failure. DESIGN Prospective case study. SETTING University-based reproductive clinics and genetics laboratory. PATIENT(S) Two hundred two infertile men presenting with severe oligozoospermia and nonobstructive azoospermia. INTERVENTION(S) Fifteen gene-specific primers were used to detect deletions of Y chromosome genes in men with spermatogenic failure. A multiplex polymerase chain reaction amplification system was developed to facilitate rapid screening. Another 24 markers for sequence-tagged sites (STS) were used to ensure the adequacy of gene-based screening. MAIN OUTCOME MEASURE(S) Detection of deletions of Y chromosome genes. RESULT(S) Of 180 patients evaluated, 19 (10.6%) had deletions of one or more genes, including DFFRY, DBY, RBM1, DAZ, CDY1, and BPY2. A second round of STS-based screenings did not show an increase in the deletion rate but more clearly defined the extent of deletion in 14 of the 19 patients. In most patients, deletions detected by gene-based screening were similar to those detected by STS markers. CONCLUSION(S) Gene-based screening with multiplex polymerase chain reaction is a rational alternative for detecting deletions of Y chromosome genes in infertile men.

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.

Successful testicular sperm extraction and paternity in an azoospermic man after bilateral postpubertal orchiopexy

Postpubertal orchiopexy is usually considered a cosmetic operation and unlikely to have any effect on fertility. We describe a 32-year-old patient with bilateral undescended testes who underwent bilateral orchiopexy at 18 years of age. He presented with primary infertility and azoospermia. After fertility counseling, testicular sperm extraction in conjunction with intracytoplasmic sperm injection was performed. A few spermatozoa were recovered and produced a fertilization rate of 42.9%. Pregnancy resulted and a healthy baby girl was delivered. We suggest that orchiopexy be recommended in infertile men with bilateral cryptorchidism, and that testicular sperm extraction be recommended if azoospermia persists after surgery.

Polymorphisms of Dioxin Receptor Complex Components and Detoxification-Related Genes Jointly Confer Susceptibility to Advanced-Stage Endometriosis in the Taiwanese Han Population

Citation Wu C‐H, Guo C‐Y, Yang J‐G, Tsai H‐D, Chang Y‐J, Tsai P‐C, Hsu C‐C, Kuo P‐L. Polymorphisms of dioxin receptor complex components and detoxification‐related genes jointly confer susceptibility to advanced‐stage endometriosis in the Taiwanese Han population. Am J Reprod Immunol 2012; 67: 160–168

SEPTIN12 Genetic Variants Confer Susceptibility to Teratozoospermia

It is estimated that 10–15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12+/+/Septin12+/− chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage. Ex vivo experiment showed truncated SEPT12 inhibits filament formation in a dose-dependent manner. This study provides the first causal link between SEPTIN12 genetic variant and male infertility with distinctive sperm pathology. Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development.

Protein tyrosine phosphatase non-receptor type 14 is a novel sperm-motility biomarker

PurposeTo understand the molecular basis of sperm-motility and to identify related novel motility biomarkers.MethodsTwo-dimensional electrophoresis (2DE) followed by Reverse-phase-nano-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) were applied to establish the human sperm proteome. Then the sperm proteome of moderate-motile human sperm fraction and that of good-motile human sperm fraction from pooled spermatozoa of forty normozoospermic donors (Group 1 subjects) were compared to identify the dysregulated proteins. Among these down-regulated proteins, Protein tyrosine phosphatase non-receptor type 14 (PTPN14) was chosen to reconfirm by Western blotting and semi-quantitative reverse transcription polymerase chain reaction. For clinical application, Western blotting and real-time reverse transcription polymerase chain reaction was performed to compare the expression level of PTPN14 in (Group 2 subjects) nine normozoospermic controls and thirty-three asthenozoospermic patients (including 21 mild asthenozoospermic cases and 12 severe cases). Finally, bioinformatic tools prediction and immunofluorescence assay were performed to elucidate the potential localization of PTPN14.ResultsThe expression levels of three proteins were observed to be lower in the moderate-motile sperm fraction than in good-motile sperm of group 1 subjects. Among three proteins with persistent down-regulation in the moderate-motile sperm, we reconfirmed that the expression level of PTPN14 was significantly lower in both mRNA and protein levels from the moderate-motile sperm fraction. Further, down-regulation of PTPN14 was found at the translational and transcriptional level in the asthenozoospermic men. Finally, Bioinformatic tools prediction and immunofluorescence assay showed that PTPN14 maybe predominantly localized at the mitochondria in the midpiece of human ejaculated sperm.ConclusionsProteomics tools were applied to identify three possible sperm motility-related proteins. Among these proteins, PTPN14 was highly likely a novel sperm-motility biomarker and a potential mitochondrial protein.

Screening of a panel of steroid-related genes showed polymorphisms of aromatase genes confer susceptibility to advanced stage endometriosis in the Taiwanese Han population

OBJECTIVE To establish a multilocus model for studying the effect of steroid-related genes on advanced stage endometriosis. MATERIALS AND METHODS A total of 121 patients with advanced stage endometriosis and 171 control women were included. Eighteen single-nucleotide polymorphisms (SNPs) from nine genes (HSD17B1, HSD17B2, HSD17B5, HSD17B6, CYP17, CYP19, ERα, ERβ, and PGR) were genotyped using the TaqMan assays. Logistic regression models were used to evaluate the genetic effects, with adjustment for other covariates. RESULTS Only the presence of the mutant CYP19 (aromatase gene) was associated with a significantly increased risk of endometriosis after adjusting for age, BMI, and parity (p = 0.002, OR = 2.69; 95% CI = 1.44-5.02). No association was ascertained between the other investigated SNPs and endometriosis. CONCLUSION Polymorphisms of the aromatase gene confer susceptibility to advanced stage endometriosis in the Taiwanese Han population.

Intermittent vaginal injections of gonadotrophins for ovarian stimulation in IVF treatment

Intermittent vaginal administration of recombinant human FSH (rhFSH), for ovarian stimulation in IVF employing the concept of uterine first-pass effect and mesotherapy, was investigated. Injection of rhFSH (437 IU, counted as six ampoules) was carried out every 3 days into the vaginal mucosa of 66 participants receiving IVF treatment between November 2004 and August 2006. The primary outcomes were number of mature oocytes, number of good grade embryos, and term live birth rate (>/=37 weeks gestation). On average, 2.94 days of injection and 16.35 ampoules of rhFSH were required to achieve proper follicular growth. Although fewer mature oocytes (5.27 +/- 3.69) were retrieved, the number of good grade embryos (3.05 +/- 1.95), number of embryos transferred (2.66 +/- 1.70), pregnancy rate per cycle started [37.9%; 95% confidence interval (CI), 27.1-49.9], implantation rate (25.5%; 95% CI, 18.0-33.0), and term live birth rate (31.8 %; 95% CI, 21.8-43.8) were comparable with conventional IVF treatments in this clinic.