A procedure to detect 6 basic STSs and 11 extended STSs in the AZF region using multiplex PCR

Microdeletions of Y chromosomes frequently occur in 3 subregions of the AZF, namely, AZFa, AZFb, and AZFc, with 6 basic STS marker sequences, which are sY84, sY86 (AZFa), sY127, sY134 (AZFb), and sY254, sY255 (AZFc). According to EAA/EMNQ guidelines, 11 additional AZFabc marker sequences should be used to determine the extent of the microdeletion in the AZF region of infertile men, which is known as 11 extended STSs. By applying mPCR, the authors develop an optimal detection procedure for the 6 basic STS and 11 extended STS using 3 multiplex PCR reactions. The first multiplex PCR reaction includes 6 basic STS plus the 2 control sequences sex-determining region Y (SRY) and zinc finger protein X/Y-linked (ZFX/Y). The second multiplex PCR reaction includes the 6 extended STS sY88, sY1182, sY105, sY121, sY1191, and sY1291 and the 2 control sequences SRY and ZFX/Y. The third multiplex PCR reaction includes the 5 extended STS sY153, sY160, sY82, sY143, and sY83 and the 2 control sequences SRY and ZFX/Y. Six basic primer sequences and eleven extended primer sequences are redesigned to simultaneously pair and amplify STS in the same multiplex reaction: set of 8 primers for 6 basic STS: 6 basic STS + 2 (SRY, ZFX/Y), 8 extension primers set E1: 6 extended STS + 2 (SRY, ZFX/Y), and 7 extension primers set E2: 5 extended STS + 2 (SRY, ZFX/Y). We successfully designed primer pairs with high specificity and stability and successfully amplified 6 basic STS and 11 extended STS, which ensures that the STSs have the correct sequence as recommended by EAA/EMQN and are consistent with the NCBI gene bank. This study has successfully developed a procedure to simultaneously detect 17 STSs, including 6 basic STSs and 11 extended STSs in the AZF region using 3 multiplex PCR reactions.

Prognostic factors for the success of obtaining spermatozoa with a biopsy of testicular tissue in men with severe forms of pathozoospermia

Приводятся данные обследования мужчин с азооспермией и олигозооспермией тяжелой степени, имеющих и не имеющих микроделеции длинного плеча Y-хромосомы, а также результаты биопсии тестикулярной ткани. Отсутствие делеций региона AZF, отсутствие гипоплазии яичек, а также нормальные показатели ФСГ, ЛГ и ингибина В являются прогностически благоприятными критериями в отношении успешности получения сперматозоидов при биопсии ткани яичка для проведения экстракорпорального оплодотворения (ЭКО). The article presents the results of a survey of men with azoospermia and severe oligozoospermia, with and without microdeletion of the long arm of the Y chromosome, as well as the results of a testicular biopsy. The absence of deletions of the AZF region, the absence of testicular hypoplasia, as well as normal levels of FSH, LH and inhibin B are prognostically favorable criteria for the success of obtaining spermatozoa with a biopsy of testicular tissue for in vitro fertilization (IVF).

Case of Inherited Partial AZFa Deletion without Impact on Male Fertility

Male factor infertility accounts for 40–50% of all infertility cases. Deletions of one or more AZF region parts in chromosome Y are one of the most common genetic causes of male infertility. Usually full or partial AZF deletions, including genes involved in spermatogenesis, are associated with spermatogenic failure. Here we report a case of a Caucasian man with partial AZFa region deletion from a couple with secondary infertility. Partial AZFa deletion, involving part of USP9Y gene appears to be benign, as we proved transmission from father to son. According to our results, it is recommended to revise guidelines on markers selected for testing of AZFa region deletion, to be more selective against DDX3Y gene and exclude probably benign microdeletions involving only USP9Y gene.

Case Report: a novel chromosomal insertion, 46, XY, inv ins(18;2)(q11.2;q13q22), in a patient with infertility and mild intellectual disability

Infertility is an important health problem affecting 15% of couples worldwide. Intellectual disability (ID) is characterized with significant impairment of intellectual function, adaptive daily life skills and social skills. Insertion is a rare chromosomal rearrangement causing infertility and ID. Here, we report a 39-year-old man presenting with primary infertility and mild ID. The patient’s spermiogram was consistent with azoospermia. Conventional cytogenetic analysis showed a novel inversion/insertion type of chromosomal aberration involving chromosomes 18 and 2: 46, XY, inv ins(18;2)(q11.2;q13q22). We carried out the array comparative genomic hybridization analysis to confirm the cytogenetic findings. Y micro-deletion analysis demonstrated that the AZF region as intact. We suggest that the novel insertion found in this case [46, XY, inv ins(18;2)(q11.2;q13q22)] may have caused infertility and mild ID in our patient. To the best of our knowledge, this chromosomal insertion has not previously been reported.

Analysis of fertility potential in men with severe azoospermia and oligospermia of various etiology

The study objective is to evaluate the significance of the Y chromosome microdeletions for prediction of spermatozoa retrieval during testicle biopsy in men with severe azoospermia and oligozoospermia.Materials and methods. In total, 109 men aged 21 to 56 years (mean age 32.7 ± 0.2 years) with infertility in marriage were examined. Cytogenetic, special andrological, spermiological, and molecular genetic examinations were performed to evaluate non-genetic and genetic causes of infertility.Results. Normal karyotype and absence of AZF deletions were found in 75 men, presence of deletions – in 34. The frequencies of pathozoospermia forms were comparable in these groups. Spermatozoa were retrieved during biopsy in 47 (62.7 %) patients without Y chromosome microdeletions and only in 7 (20.6 %) patients with Y chromosome microdeletions. The men with AZF deletions were divided into 2 subgroups: men with complete AZF region deletions (n = 25) and men with partial AZF deletions (n = 9). Among men with complete deletions, azoospermia was diagnosed in 25 (100 %), spermatozoa were retrieved during biopsy in 2 (8 %); among men with partial deletions, azoospermia was diagnosed in 7 (77.8 %), severe oligozoospermia in 2 (22.2 %), spermatozoa were retrieved during biopsy in 5 (56 %). Then the patients were divided according to another criterion: 54 patients from whom spermatozoa were retrieved during biopsy and 55 men with negative results. Among patients with successful result of biopsy, Y chromosome microdeletions were identified in 7 (13 %); among patient with negative biopsy result – in 27 (49 %) (р < 0.01).Conclusion. Success rate of spermatozoa retrieval during testicle biopsy is significantly higher in men without AZF deletions (р < 0.01) than in men with deletions. Molecular genetic examination of Y chromosome microdeletions is recommended for men with azoospermia and severe oligozoospermia because it allows diagnosing of cause male infertility and predicting.

Clinical, Hormonal, and Genetic Evaluation of Idiopathic Nonobstructive Azoospermia and Klinefelter Syndrome Patients

To investigate the clinical, hormonal, and genetic factors in infertile men with idiopathic nonobstructive azoospermia (NOA) or azoospermic Klinefelter syndrome (KFS), a total of 556 and 96 patients, respectively, were included in this study. All patient samples were analyzed cytogenetically. Serum reproductive hormone levels were measured. Microdeletions in the azoospermia factor (AZF) region of the Y chromosome were detected by multiplex PCR using 16 specific sequence-tagged sites. FSH and LH levels in both NOA and KFS patients were significantly higher than the normal range, and the testosterone level in KFS patients was significantly lower. Ninety-two (95.8%) of the KFS patients showed non-mosaic 47,XXY karyotypes and 47,XXY,inv(9)(p11.1q13); the other KFS patients had mosaic karyotypes of 47,XXY/46,XY, 47,XXY/46,XX, and 47,XXY/48,XXXY/46,XX. Among the 556 idiopathic NOA patients with normal karyotypes, 67 (12.05%) had microdeletions in the AZF region of the Y chromosome. Microdeletions were most frequently detected in the AZFc region, followed by AZFa, AZFb, AZFbc, and partial AZFc deletions. However, Y chromosome microdeletions were not found in any of the azoospermic KFS patients. In view of the hormonal and genetic abnormalities in infertile men with idiopathic NOA and with azoospermic KFS, genetic testing for karyotype, Y chromosome microdeletions, and hormonal parameters is advocated.