Cisplatin-induced azoospermia and testicular damage ameliorated by adipose-derived mesenchymal stem cells

BACKGROUND: The testes are highly susceptible to the adverse effects of chemotherapy and radiation at all stages of life. Exposure to these threats mainly occurs during cancer treatment and as an occupational hazard in radiation centers. The present study investigated the regenerative ability of adipose-derived mesenchymal stem cells (ADMSCs) against the adverse effects of cisplatin on the structure and function of the testes. METHODS: New Zealand white rabbits (N = 15) were divided into three groups of five: a negative control group (no treatment), a cisplatin group (single dose of cisplatin into each testis followed three days later by a PBS injection), and a cisplatin + ADMSCs group (cisplatin injection followed three days later by an ADMSC injection). On day 45 post-treatment, serum testosterone levels were evaluated, and the testes and epididymis were collected for histology, oxidative stress examination, and epididymal sperm analysis. RESULTS: Cisplatin caused damage to the testicular tissue and decreased serum testosterone levels, epididymal sperm counts, and oxidants. An antioxidant imbalance was detected due to increasing malondialdehyde (MDA) and reduced glutathione (GSH) levels in testicular tissue. The ADMSC-treated group displayed a moderate epididymal sperm count, adequate antioxidant protection, suitable hormone levels, and enhanced testicular tissue morphology. CONCLUSIONS: ADMSCs treatment repaired damaged testicular tissue, enhanced biochemical parameters, and modified pathological changes caused by cisplatin.

Identification of risk genes in Chinese nonobstructive azoospermia patients based on whole-exome sequencing / 亚洲男科学杂志(英文版)

Nonobstructive azoospermia (NOA) is a severe condition in infertile men, and increasing numbers of causative genes have been identified during the last few decades. Although certain causative genes can explain the presence of NOA in some patients, a proportion of NOA patients remain to be addressed. This study aimed to investigate potential high-risk genes associated with spermatogenesis in idiopathic NOA patients by whole-exome sequencing. Whole-exome sequencing was performed in 46 male patients diagnosed with NOA. First, screening was performed for 119 genes known to be related to male infertility. Next, further screening was performed to determine potential high-risk causative genes for NOA by comparisons with 68 healthy male controls. Finally, risk genes with high/specific expression in the testes were selected and their expression fluctuations during spermatogenesis were graphed. The frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene pathogenic variant carriers was higher in the NOA patients compared with the healthy controls. Potential risk genes that may be causes of NOA were identified, including seven genes that were highly/specifically expressed in the testes. Four risk genes previously reported to be involved in spermatogenesis (MutS homolog 5 [MSH5], cilia- and flagella-associated protein 54 [CFAP54], MAP7 domain containing 3 [MAP7D3], and coiled-coil domain containing 33 [CCDC33]) and three novel risk genes (coiled-coil domain containing 168 [CCDC168], chromosome 16 open reading frame 96 [C16orf96], and serine protease 48 [PRSS48]) were identified to be highly or specifically expressed in the testes and significantly different in the 46 NOA patients compared with 68 healthy controls. This study on clinical NOA patients provides further evidence for the four previously reported risk genes. The present findings pave the way for further functional investigations and provide candidate risk genes for genetic diagnosis of NOA.

Evaluación de la eficacia de los laboratorios de embriología y anatomía patológica en el análisis de las biopsias testiculares de pacientes azoospérmicos / Evaluation of the Efficacy of the Laboratories of Embryology and Pathological Anatomy in the Analysis of Testicular Biopsies of Patients with Azoospermia

Objetivos: El propósito de este estudio es evaluar la eficacia de los laboratorios de embriología y de anatomía patológica para hallar espermatozoides en las muestras de tejido testicular obtenido por biopsia testicular (testicular sperm extraction, TESE) en pacientes con azoospermia no obstructiva. Materiales y métodos: Se realizó un análisis retrospectivo y prospectivo de todos los pacientes con azoospermia no obstructiva atendidos en CRECER y en la Clínica Privada Pueyrredón, entre enero de 2006 y diciembre de 2016. En este estudio solo se incluyeron aquellos pacientes en los que la muestra obtenida con TESE fue enviada simultáneamente al anatomopatólogo y al laboratorio de embriología. Para el análisis de los resultados de las biopsias el estudio se detuvo a fines de 2016, pero el seguimiento de los pacientes continuó hasta el mes de octubre de 2017, registrándose todos aquellos casos que realizaron procedimientos de inyección intracitoplasmática de espermatozoides (intracytoplasmic sperm injection, ICSI) con muestras obtenidas de TESE y se anotó la obtención de embriones, embarazos y nacimientos. Resultados: El laboratorio de embriología halló espermatozoides en 36 de los 68 pacientes (52,9%), mientras que el laboratorio de patología solo informó presencia en 21 pacientes (30,88%). Hubo acuerdo en el hallazgo de espermatozoides entre ambos laboratorios en 20 de los 68 casos (29,41%), mientras que en 16 pacientes el laboratorio de embriología encontró espermatozoides donde el de patología no pudo hacerlo (23,53%). Al mismo tiempo, el laboratorio de patología halló espermatozoides solo en un caso en el que el de embriología informó su ausencia para la misma muestra analizada (1,47%) (p=0,0003). Conclusiones: El laboratorio de embriología es significativamente más eficaz para determinar la presencia de espermatozoides en las muestras de TESE, teniendo mejor rendimiento que el de patología, por lo que consideramos que, si las muestras fueran analizadas solo por el patólogo, se perdería la posibilidad de lograr muchos embarazos realizando ICSI más TESE.(AU)

Objectives: The purpose of this study is to evaluate the efficacy of embryology and pathological anatomy laboratories to find spermatozoa in testicular tissue samples obtained by testicular sperm extraction (TESE) in patients with non-obstructive azoospermia. Materials and methods: It was carried out a retrospective and prospective analysis of all the patients with non-obstructive azoospermia treated at CRECER and at Clínica Privada Pueyrredón, between January 2006 and December 2016. This study only includes patients in whom the sample obtained with TESE was sent at the same time to the pathology and embryology laboratory. For the analysis of the results of the biopsies, the study was stopped at the end of 2016, but the follow-up of the patients continued until October 2017, registering all those cases that performed intracytoplasmic sperm injection (ICSI) with samples obtained from TESE and wrote down the patients who´ve got embryos, pregnancies, and births. Results: The embryology laboratory found sperm in 36 of the 68 patients (52.9%), while the pathology laboratory only reported presence in 21 patients (30.88%). There was agreement in the finding of sperm between both laboratories in 20 of the 68 cases (29.41%), while in 16 patients the embryology laboratory found sperm where the pathology department could not do so (23.53%). At the same time, the pathology laboratory found sperm only in one case in which the embryology department reported its absence for the same sample analyzed (1.47%) (p=0.0003). Conclusions: The embryology laboratory is significantly more efficient to determine the presence of sperm in the samples of TESE, having better performance than the pathology one. Taking into account that, we believe that if the samples are only analyzed by the pathologist, the possibility of getting many pregnancies performing ICSI plus TESE would be lost. (AU)

Azoospermia y fertilización asistida: rol de la histología testicular en el éxito de icsi / Azoospermia and assisted reproduction: role of testicular histology in the success of ICSI

Introducción: La infertilidad masculina afecta aproximadamente al 7por ciento de los hombres, presentándose hasta el 15 por ciento de ellos con azoospermia. El conocimiento del tipo de azoospermia (obstructiva o no obstructiva) y la localización de la falla (pre-testicular, testicular o post-testicular) es vital para conocer el pronóstico de fertilidad de la pareja y plantear un plan terapéutico adecuado. Actualmente, la extracción de espermatozoides desde epidídimo o testículo de pacientes azoospérmicos, y la posterior inyección intracitoplásmática de éstos (ICSI, por sus siglas en inglés) ha permitido obtener embriones viables para su posterior transferencia. Materiales y métodos: Estudio descriptivo retrospectivo de 42 parejas infértiles con diagnóstico de azoospermia; que se sometieron a biopsia testicular, ICSI y posterior transferencia de embriones, entre los años 2004 y 2012. Se lleva a cabo un análisis de la edad de los pacientes, resultados de la histopatología testicular y su asociación con los resultados de la fertilización asistida. Resultados: 42 pacientes azoospérmicos se sometieron a biopsia testicular y extracción de espermatozoides en el mismo acto quirúrgico. La edad promedio de los pacientes fue de 36 años para los hombres y 32 años para las mujeres. En el análisis histológico de los tejidos testiculares, el 31por ciento de los pacientes presentaban espermatogénesis conservada (EC), el 35.7 por ciento atrofia mixta (AM), el 14.3 por ciento hipoespermatogénesis (HE), el 14.3 por ciento detención de la maduración (DM) y un 4.8 por ciento presentaba otras histologías. Lograron embarazo clínico 14 de 42 parejas (33,3 por ciento), siendo la tasa de embarazo específica por patología de 38,5 por ciento para EC, 26.7 por ciento para AM, 50 por ciento para HE, 16,7 por ciento para DM y 50 por ciento para las otras histologías. 12 de las 42 parejas realizaron más de un ciclo de transferencias...

Introduction: Male infertility affects approximately 7percent of men, presenting up to 15 percent with azoospermia. Knowing the type of azoospermia (obstructive or non-obstructive) and the location of the problem (pre-testicular, testicular and post-testicular) is vital to recognize the fertility prognosis of the couple and present a proper treatment plan. Currently, the extraction of sperm from epididymis or testis of azoospermic patients, and subsequent intracytoplasmic sperm injection (ICSI) has yielded viable embryos for transfer. Materials and Methods: Retrospective study of 42 infertile couples diagnosed with azoospermia, who underwent testicular biopsy, ICSI and subsequent embryo transfer, between 2004 and 2012. We performed an analysis of the patients’ age, testicular histopathology results and their association with assisted fertilization outcome. Results: 42 azoospermic patients underwent testicular biopsy and sperm extraction in the same surgery. The average age of patients was 36 years for men and 32 years for women. Histologic analysis of testicular tissue showed that 31 percent of patients had normal spermatogenesis (NS), 35.7 percent mixed atrophy (MA), 14.3 percent hypospermatogenesis (HS), 14.3 percent maturation arrest (MTA) and 4.8 percent had other histologies. 14 of 42 couples achieved clinical pregnancy (33.3 percent), with a specific pregnancy rate of 38.5 percent for NS, 26.7 percent for MA, 50 percent for HS, 16.7 percent for MTA and 50 percent for other histologies. 12 of 42 couples underwent more than one transfer cycle. Conclusions: The use of ICSI is a suitable alternative for infertile couples with severe male factor. The results of this technique are favorable for most histologies. Patients with MA exhibit sertoli solo syndrome and / or tubular sclerosis foci associated to regions with normal spermatogenesis, enabling the sperm extraction for ICSI.

Sperm retrieval techniques for assisted reproduction

Different surgical methods such as PESA, MESA, TESA, TESE and micro-TESE have been developed to retrieve spermatozoa from either the epididymis or the testis according to the type of azoospermia, i.e., obstructive or non-obstructive. Laboratory techniques are used to remove contaminants, cellular debris, and red blood cells following collection of the epididymal fluid or testicular tissue. Surgically-retrieved spermatozoa may be used for intracytoplasmic sperm injection (ICSI) and/or cryopreservation. In this article, we review the surgical procedures for retrieving spermatozoa from both the epididymis and the testicle and provide technical details of the commonly used methods. A critical analysis of the advantages and limitations of the current surgical methods to retrieve sperm from males with obstructive and non-obstructive azoospermia is presented along with an overview of the laboratory techniques routinely used to process surgically-retrieved sperm. Lastly, we summarize the results from the current literature of sperm retrieval, as well as the clinical outcome of ICSI in the clinical scenario of obstructive and non-obstructive azoospermia.

Alteración de la fertilidad masculina por hiperplasia suprarrenal congénita: Azoospermia reversible con terapia de glucocorticoide / Congenital adrenal hyperplasia causing male infertility: Report of one case

In males, congenital adrenal hyperplasia due to 21 hydroxylase deficiency is associated to normal fertility or infertility caused by a hypogonadotrophic hypogonadism (HH) or gonadal damage caused by intratesticular adrenal remnants. We report a 29-year-old male with azoospermia, without any important personal or family background. Physical examination was normal, his height was 150 cm and his testicular volume was 10 ml (normal 15 to 25 ml). Laboratory showed a normal testosterone and FSH and LH in the low normal limit. These results discarded a HH, whose diagnostic requirements are a low testosterone and inadequately normal or low gonadotrophins. A testicular biopsy was informed as compatible with HH. A 21 hydroxylase deficiency was suspected and confirmed with extremely high levels of 17 hydroxyprogesterone at baseline and after stimulation with fast acting ACTH. Clomiphene citrate did not increase testosterone or gonatrophin levels. Testicular ultrasound discarded the presence of adrenal nodules. Betametasone therapy resulted in a normal testicular development, normalization of sperm count, reduction of 17 hydroxyprogesterone and testosterone levels with an ulterior rise of the latter. Spontaneous paternity was achieved twice. It must be remembered that in cases of azoospermia due to congenital adrenal hyperplasia, testosterone produced by adrenal glands hinders the laboratory diagnosis of HH.

Chronology of meiosis & synaptonemal complex abnormalities in normal & abnormal spermatogenesis.

Background & objectives: The study was taken up to define criteria of normality for meiosis by assessing the frequency of meiotic prophase cell types, the frequency of pachytene substage in normal and abnormal spermatogenesis and to determine what synaptonemal complex. Methods: A quantitative and qualitative analysis of the first meiotic prophase was performed in 10 patients presenting with non-obstructive infertility and 10 controls, using dual colour immunocytochemistry with SCP3 and BRCA1 which visualise axial elements and synaptonemal complexes (SC). The respective frequencies of the leptotene, zygotene and pachytene stages as well as the frequencies of the four substages of pachytene were evaluated. The frequencies of the main types of meiotic abnormalities at pachytene were also assessed. Results: The frequencies of leptotene and zygotene stages were significantly higher in patients (7.95 and 9.75%) than in controls (2.30 and 1.45%), whereas the frequency of pachytene was significantly higher in controls than in patients (96.25 vs. 75.30%). Detailed analysis of the sex chromosomes revealed that the controls showed a presence of late pachytene substages (P3 + P4 = 64.40%), whereas the patients showed a early pachytene substages (P1 + P2 = 63.40%). From these results, a new index was defined to evaluate spermatogenesis: the Pachytene Index, or PI (PI = P1 + P2 / P1 + P2 + P3 + P4). The same abnormalities (asynapsis, fragmented SC, dotted SC, thin SC) were observed in controls and in patients, but with different frequencies. The most frequent abnormality was fragmented SC, with a significant difference between patients and controls (15.28 vs. 9.74%). There was a significant difference between patients and controls for the frequency of asynapsed nuclei (7.97 vs. 2.95%) while the difference in other abnormalities were not significant. Interpretation & conclusion: The accumulation of early primary spermatocytes is an indication that progression of meiosis is defective in spermatogenesis failures. The value of the PI less than 0.50 indicates that the kinetic of meiosis is normal at pachytene. There is no normal spermatogenesis when the frequency of one or several SC abnormalities is significantly higher than in controls and/ or when the PI is more than 0.50.

Role of testicular biopsy in the evaluation of azoospermic men

A total of 67 infertile men with azoospermia were studied over 5 years period [February 1997-March 2002]. Their mean age was 33 years and mean duration of infertility was 6 years. They underwent bilateral testicular biopsy under general anaesthesia and the specimens were categorizwd according to histopathological patterns. These included normal spermatgenesis 12 [17.9%]; hypospermatogenesis 4 [5.9%]; spermatogenic arrest 39 [58.2%]; sertoli cell only 7 [10.4%]; and complete tubular hyalinization 5 [7.4%]. This study highlights the role of testicular biopsy as a reliable and useful technique for the investigation of patients with azoospermia

[Evaluation of Spermatogenesis in non-obstructive azoospermic patients with histopathological and cytological methods]

The ability to use only a few spermatozoa using testicular biopsy and by microinjection technique to achieve fertilization and pregnancy has revolutionized the potentials to treat patients suffering from azoospermia. However, spermatogenesis is defective in men with non-obstructive azoospermia [NOA] resulting in failure to detect spermatozoa. In order to achieve a more sensitive and reliable method for detecting sperm/spermatid in testes of NOA patients, we compared histopathological and cytological methods. Eighty six NOA patients were included in a prospective study. History taking, physical examination and hormonal profile [including FSH] were done initially. Thirty six patients had testis pathology report before enrolling in to our study. The patients underwent multiple bilateral testis biopsies until successful retrieval of sperm/spermatid. Half of each biopsy specimen underwent cytological evaluation [mechanical and enzymatic] and the other half was sent for pathological evaluation [TBX]. The male and female mean ages were 37 [25-59] and 32 [23-42] years, respectively. The mean infertility duration was 7 years. The mean volume of right and left testis were 16.4 and 16.2 ml, respectively. Mean FSH level was 18.1 +/- 14.2 ml U/ml. In cytology, sperms and spermatids were seen in 65 and 18 patients, respectively and in pathology slides in 51 and 16 patients, respectively. In our study, the relationship between visualizing testicular sperm/spermatid and TESE had sensitivity of 80% and negative predictive value of 15%. Sixty one patients had sufficient number of sperm/spermatid for ICSI and with this treatment fifty seven embryos were transferred and seven clinical pregnancies were observed. In conclusion, in men with non-obstructive azoospermia, TESE is more sensitive and reliable than histopathology evaluation. This means that TESE may help in deciding for treatment of severe male factor infertility, even when histopathologic examination is inconclusive