Extreme Oligozoospermia Research Articles

Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience.

Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended. To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study). In the validation study, ES was performed on DNA of patients already diagnosed with AZF deletions (n=17), (non-)mosaic sex chromosomal aneuploidies or structural chromosomal anomalies (n=37), CFTR variants (n=26), or variants in known infertility genes (n=4), and 90 controls. The data were analyzed using our standard diagnostic pipeline, with a bioinformatic filter for 130 male infertility genes. In the evaluation study, results of 292 clinical exomes were included. All previously reported variants in the validation cohort, including clinically relevant Y-chromosomal microdeletions, were correctly identified and reliably detected. In the evaluation study, we identified one or more clinically relevant genetic anomalies in 67 of 292 of all cases (22.9%): these included aberrations that could have been detected with current methods in 30 of 67 patients (10.2% of total), (possible) (mono)genetic causes in the male infertility gene panel in 28 of 67 patients (9.6%), and carriership of cystic fibrosis in nine of 67 patients (3.1%). ES is a reliable first-tier method to detect the most common genetic causes of male infertility and, as additional genetic causes can be detected, in our evaluation cohort the diagnostic yield almost doubled (10.2%-19.8%, excluding CF carriers). A genetic diagnosis provides answers on the cause of infertility and helps the professionals in the counseling for treatment, possible co-morbidities and risk for offspring and/or family members. Karyotyping will still remain necessary for detecting balanced translocations or low-grade chromosomal mosaicism.

Treatment progress of cryptozoospermia with Western Medicine and traditional Chinese medicine: A literature review.

Cryptozoospermia is an extreme oligozoospermia with an unsatisfactory treatment effect, with an incidence rate of approximately 8.73% in male infertility, whose effective solution has become the call of the times. Western Medicine has achieved certain effects through drugs, surgery, and assisted reproductive therapy, but this is still not ideal. Traditional Chinese medicine (TCM) has made many achievements in other disciplines; however, there is still a lack of evidence-based medical evidence to improve sperm production. The relevant literatures from the China National Knowledge Internet (CNKI) and PubMed in the past 10 years were collected in this article, of which the mechanisms, advantages, or current controversies of various treatment methods of Western Medicine and TCM were analyzed, to find new treatment methods and research directions. With the development of modern science and technology, medical treatments for cryptozoospermia have become increasingly abundant; however, there is still no universally recognized unified and effective guiding plan. Although TCM has not been fully verified by evidence-based medicine, most TCM combined with Western Medicine can achieve unexpected results. The combination of TCM and Western Medicine may become a bane for cryptozoospermia and bring good news to infertile men worldwide.

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.

Sequential interval micro-droplet loading in closed hemi-straw carrier system: A convenient and efficient method for ultra-rapid cryopreservation in extreme oligozoospermia

Cryopreservation of human spermatozoa with low concentration while maintaining adequate post-thawing motility remains a major challenge for male fertility preservation. A convenient and efficient ultra-rapid freezing method for small amounts of human spermatozoa in a closed Hemi-Straw carrier system (CHS) was developed. Spermatozoa from 60 healthy men were involved in a parameter refining test and another 15 extreme oligozoospermic specimens were assigned to a verification test. A commercialized sperm freezing medium, Quinn's Advantage® Sperm Freeze medium (glycerol and sucrose as the cryoprotective agent) was used in the study. The results showed that the highest recovery rates would be obtained via the method of 2 μl single droplet sequential interval loading, by placing the straw at 1 cm above the liquid nitrogen (LN2) surface for 60 s during freezing and 2 cm above the LN2 for 2 min during thawing. This method was applied in cryopreservation for the normozoospermic specimens and compared with a conventional slow freezing method. The results were better than those in the control group in the total motility recovery rate (77.8 ± 11.2% vs 56.6 ± 11.9%, P < 0.01), progressive motility recovery rate (77.6 ± 13.2% vs 47.7 ± 14.6%, P < 0.01), 24 h survival index (60.9 ± 13.4% vs 42.1 ± 14.1%, P < 0.01) and the sperm DNA fragment index (4.2 ± 3.7% vs 5.8 ± 3.7%, P = 0.126). This method was applied to the oligozoospermic specimens. Motile spermatozoa could be found in 12 of 15 cases in the ultra-rapid freezing group, while only in 7 cases in control group. The results indicated that this freezing method was simple, convenient and bio-safe for cryopreservation of severe oligozoospermic specimens.

Extreme spermatogenesis failure: andrological phenotype and intracytoplasmic sperm injection outcomes

Patients with very low sperm count through direct sperm examination can exhibit extreme oligozoospermia or cryptozoospermia (after centrifugation). The management of these patients is a real challenge for both clinicians and biologists. In this retrospective and comparative cohort study, we compared the andrological phenotype of patients with extreme alterations of spermatogenesis and assessed whether the origin of spermatozoa (testicular or ejaculate) had any influence on intracytoplasmic sperm injection (ICSI) outcomes. A total of 161 ICSI cycles were performed using ejaculated spermatozoa from 75 patients with extreme oligozoospermia (EOS) or cryptozoospermia (CS) and 150 ICSI cycles using extracted testicular spermatozoa from 74 patients with non-obstructive azoospermia (NOA). Physical, hormonal, ultrasound assessments, and ICSI outcomes were performed in each group. Cryptorchidism was significantly more frequent in the NOA group (60.8% vs. 22.6%, p=0.001). FSH levels were significantly higher [18.9IU/L (5.9-27.0) vs. 15.3IU/L (9.0-46.5), p=0.001] and the majority of inhibin B levels measured were found mostly undetectable in the NOA group as compared to EOS/CS group (31.1% vs. 10.7%, p=0.0004). Moreover, we found no significant differences in the respect to the fertilization rates (48.9% and 43.3%, p=0.43), implantation rates (17.4% and 15.9%, p=0.77), and percentage of top quality embryo (22.4% and 20.4%, p=0.73) between the two groups. The clinical pregnancy rates per embryo transferred were comparable in both groups (28.3% and 27.4%, p=0.89). In this study, we showed for the first time a different andrological phenotype between EOS/CS and NOA groups. Indeed, cryptorchidism was significantly more frequent with more severe endocrine parameters found in the NOA group. These results reflect a more profound alteration in spermatogenesis in NOA patients. However, there was no difference in ICSI outcomes between NOA and EOS/CS groups.

The value of chromosomal analysis in oligozoospermic men

To determine the prevalence of chromosomal abnormalities in relation to sperm concentration in subfertile oligozoospermic men. Retrospective cohort study. Two teaching hospitals. We retrospectively studied all men who received chromosomal analysis prior to intracytoplasmic sperm injection (ICSI) treatment from 2000 to 2010 in two teaching hospitals. None. The results of chromosomal analysis and semen analysis were recorded. The frequency of abnormal karyotypes was analyzed in relation to the sperm concentration, categorized as extreme oligozoospermia (>0 to ≤1 million/mL), severe oligozoospermia (>1 to ≤5 million/mL), moderate oligozoospermia (>5 to ≤20 million/mL), or normospermia (>20 million/mL). Among 582 male ICSI candidates, the rates of abnormal karyotypes were 1.2% (2/162), 2.2% (5/227), and 1.5% (2/130) for men with extreme, severe, and moderate oligozoospermia, respectively. No abnormalities were present in normospermic men. The risk of conceiving a viable child with unbalanced structural chromosomal abnormalities in men with oligozoospermia may not justify karyotyping.

Performance of testicular versus ejaculated spermatozoa in men with extreme oligozoospermia

Performance of testicular versus ejaculated spermatozoa in men with extreme oligozoospermia

“One sperm, one embryo, one baby”?

“One sperm, one embryo, one baby”?

Enhanced serum prolactin concentration after metoclopramide stimulation in idiopathic oligozoospermia and azoospermia.

The aim of the study was to evaluate the hormonal pattern in men with idiopathic oligozoospermia and azoospermia. Material consisted of 13 sterile men, 21-34 years old (the mean - 27 years). Three of 13 patients had the third degree oligozoospermia, 3 - extreme oligozoospermia and 7 - azoospermia. The control group consisted of 11 healthy men who were found to have normozoospermia. In all patients and in control subjects the estimation of LH, FSH, prolactin (PRL), testosterone and dihydrotestosterone (DHT) in basal conditions and also PRL determination at 15, 30, 60 min after stimulation with metoclopramide (MCP 10 mg i.v.) were performed by radioimmunoassay. The results indicate that men with idiopathic oligo- or azoospermia have significantly higher prolactin level in response to dopaminergic receptor blockade with metoclopramide and significantly lower serum testosterone and DHT concentrations.

Clinical data and parenthood of 63 infertile and Y-microdeleted men

To collect follow-up data for infertile men with Y microdeletion. Retrospective, observational survey. Multicenter IVF units associated with genetics laboratories. Sixty-three patients with Y microdeletion. Karyotype analysis, Y microdeletion screening, and assisted reproductive technology. Medical history, karyotype, nature of the AZF deletion, semen parameters, testis biopsy results, choice of assisted reproductive technology, and results of intracytoplasmic sperm injection (ICSI). Abnormal karyotypes were found in 8 men (12.7%), who were azoospermic except 1. Of these 8 men, 5 presented a combined AZFb+c deletion, and 3 had a deletion in AZFc only. Most men (39 of 63) were azoospermic, 3 were cryptoazoospermic, and 19 had extreme oligozoospermia (sperm concentration </=1.10(6)/mL). Sperm concentration above 1.10(6)/mL was found for 2 men (3.1%). A testis biopsy was performed in 27 azoospermic men, resulting in positive sperm extraction in 6 cases. To date, 42 ICSI cycles with either testicular (n = 5) or ejaculated spermatozoa (n = 37) have been carried out in 23 couples with male partners with AZFc deletion. Eighteen clinical pregnancies were obtained, leading to the birth of 14 babies. Donor insemination had been chosen by 28 couples, leading to the birth of 9 children. Karyotype analysis should be systematically performed in Y microdeleted men. Intracytoplasmic sperm injection can be offered to half of AZFc-deleted patients, providing real opportunities to have a child.

Optimal management of extreme oligozoospermia by an appropriate cryopreservation programme

Severe oligozoospermia is characterized by sperm count fluctuations that may result in insufficient quantities of motile sperm for ICSI on the day of oocyte retrieval, thus necessitating testicular biopsy. To avoid this, we proposed that patients, with transient azoospermia or repeatedly low sperm counts, make a safety pool of frozen spermatozoa before ICSI attempts. Seventy cryptozoospermic (<10(3) spermatozoa/ml) and 46 oligozoospermic patients (10(3)-10(5)/ml) were included. Although all oligozoospermic patients succeeded in sperm banking, only 44 of 70 cryptozoospermic patients were successful. Others underwent testicular extraction of spermatozoa. The ICSI results for frozen sperm from cryptozoospermic patients were compared with those obtained with fresh sperm from a group of normal patients (>10(5) spermatozoa/ml). In this prospective matched, controlled study, five cryptozoospermic, but no oligozoospermic, patients failed to produce sperm on the ICSI day, and frozen sperm was used instead. Although fertilization and pregnancy rates (per attempt) using fresh (49% and 5/44, respectively) and frozen sperm (54% and one-fifth, respectively) were similar for this cryptozoospermic group, the results for fresh sperm were significantly lower when compared with the control group (66% and 16/43, P < 0.0001, P < 0.001, respectively). In contrast, results for the oligospermic and control groups were similar. Banking of ejaculated sperm is helpful for cryptozoospermic patients.

Collecting Human Spermatozoa onto Filters for FISH

To describe a simple method of fluorescence in situ hybridization (FISH) performed on germ cells collected on filters that allows the study of aneuploid sperm frequency in cases of extreme oligozoospermia. The aneuploidy rates for chromosomes X, Y and 18 were analyzed in three groups to test the reliability of the filtration-FISH procedure when compared to controls and to evaluate the frequency of aneuploid spermatozoa in five oligospermic men. Filtration of semen samples before FISH does not modify the basal rate of aneuploidy even after various sperm dilutions. In oligospermic men, the frequency of gonosomal aneuploidies is significantly increased. The filtration-FISH technique is a powerful method of analyzing chromosomal abnormalities in sperm nuclei in men with extreme oligozoospermia.

Genetic risk factors in infertile men with severe oligozoospermia and azoospermia

Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic risk factors. In this study 150 men from couples requesting ICSI were investigated for genetic abnormalities, such as constitutive chromosome abnormalities, microdeletions of the Y chromosome (AZF region) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genetic analysis identified 16/150 (10.6%) abnormal karyotypes, 8/150 (5.3%) AZFc deletions and 14/150 (9.3%) CFTR gene mutations. An abnormal karyotype was found both in men with oligozoospermia and azoospermia: 9 men had a sex-chromosomal aneuploidy, 6 translocations were identified and one marker chromosome was found. Y chromosomal microdeletions were mainly associated with male infertility, due to testicular insufficiency. All deletions identified comprised the AZFc region, containing the Deleted in Azoospermia (DAZ) gene. CFTR gene mutations were commonly seen in men with congenital absence of the vas deferens, but also in 16% of men with azoospermia without any apparent abnormality of the vas deferens. A genetic abnormality was identified in 36/150 (24%) men with extreme oligozoospermia and azoospermia. Application of ICSI in these couples can result in offspring with an enhanced risk of unbalanced chromosome complement, male infertility due to the transmission of a Y-chromosomal microdeletion, and cystic fibrosis if both partners are CFTR gene mutation carriers. Genetic testing and counselling is clearly indicated for these couples before ICSI is considered.

Validation of a simple Yq deletion screening programme in an ICSI candidate population.

This study reports on the validation of a diagnostic screening programme for Yq deletions in a population of infertile men. First, an unselected group of 402 intracytoplasmic sperm injection (ICSI) candidate patients was screened prospectively by means of three polymerase chain reactions (PCR) each with one marker in the region AZFa, AZFb or AZFc. With this screening strategy, eight males (2.2%) were found to carry a deletion in Yq11. Secondly, a subgroup of males were further analysed by multiplex PCR with 27 sequence-tagged sites. In this group of 229 cytogenetically normal males with azoospermia, cryptozoospermia or extreme oligozoospermia, including some patients with varicocele or a history of cryptorchidism, only one additional microdeleted patient was found with the multiplex PCR. Hence we obtained a frequency of 2.2% (9/402) or 4% (9/229) in the unselected and selected patient groups respectively. We conclude that in a diagnostic programme for Yq deletions in ICSI candidates it might be sufficient to use only four markers representing the three AZF regions and a more distal region in AZFc. In this way, it is possible to detect most, if not all, Yq deletions which might be the causal factor in the patient's infertility.

MALE FERTILITY DISORDERS

Effective treatments for extreme oligozoospermia include in vitro fertilization, recovery of sperm through epididymal sperm aspiration or testicular sperm aspiration, and direct injection of sperm through intracytoplasmic sperm injection. The clinical evaluation of and treatment options for male patients with fertility disorders are detailed in this article.

Evidence of disturbed meiosis in a man referred for intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has become the treatment of choice in male infertility due to extreme oligozoospermia. Initially, the technique was limited to the selection and injection of motile spermatozoa with normal morphology; although it has now been shown that spermatozoa with abnormal morphology can also be used successfully.1 We describe a patient referred for ICSI who presented with phenotypically abnormal sperm and we show that in this patient the disturbed spermatogenesis was associated with chromosome abnormalities and abnormal meiosis.

Genetic Counseling in a Patient with XXY/XXXY/XY Mosaic Klinefelter’s Syndrome: Estimate of Sex Chromosome Aberrations in Sperm Before Intracytoplasmic Sperm Injection

Objective: To report the sex chromosome aberrations in the sperm of a patient with mosaic Klinefelter’s syndrome before ICSI. Design: Case report. Setting: Institute of Human Genetics, University Hospital Patient(s): A patient with an XXY/XXXY/XY mosaic Klinefelter’s syndrome and extreme oligozoospermia. Intervention(s): Skin biopsy, buccal smear, hair root sampling, and semen sampling. Main Outcome Measure(s): The karyotypes of three additional somatic cell systems and the ratio of sex chromosome aberrations in sperm. Result(s): After two-color fluorescence in situ hybridization of 202 interphase sperm nuclei, both the proportion of hyperhaploid 24,XY and 25,XXY sperm (5.0% and 0.5%, respectively) and of hyperhaploid 24,XX sperm (2.0%) were elevated. In contrast with peripheral lymphocytes, 93.9% of which showed sex chromosome aberrations, in the present patient only 7.5% of sperm proved to be hyperhaploid with an extra sex chromosome. Conclusion(s): The determination of sex chromosome aberrations in the sperm of a patient with mosaic Klinefelter’s syndrome may provide additional information to estimate the transmission risk to his offspring.

Micromanipulation of gametes and embryos: Cryopreservation of a single human spermatozoon within an isolated zona pellucida.

There are several reasons for a need to cryostore a smallnumber of spermatozoa. Men who require testicular biopsybenefit tremendously if the presence of spermatozoa isknown before the onset of follicular recruitment in thepartner. This can usually be achieved by cryopreserving thebiopsy material or suspension of cells. But post-thawsuccess depends on the presence of relatively largenumbers of spermatozoa, since the addition of cryo-protectant requires removal by centrifugation whichdestroys many viable cells. The cut-off level forconventional freezing in mini-straws is 1200 spermatozoa,but many men have even less cells. Freezing of multiplesamples of very few spermatozoa would neverthelessguarantee several attempts at intracytoplasmic sperminjection (ICSI) and avoid repeated testicular aspirations incase further treatments are required.The recent of use of testicular spermatozoa has greatlyemphasized the need for the storage of aspirated testiculartissue or of spermatozoa that are recovered from suchtissue (Salzbrun et al ., 1996). Other methods of cryostoringsmall numbers of spermatozoa retrieved from the testisinclude the use of small droplets of medium which areplaced on a dry ice surface before freezing in liquidnitrogen, to give pregnancy rates similar to those obtainedwith fresh testicular spermatozoa (Gil-Salom et al ., 1996).Though both techniques are elegant and extremely useful,application falters when levels drop below one thousand.Special methods are necessary to store spermatozoa incases of extreme oligozoospermia. This circumstancearises constantly during ICSI when epididymal ortesticular spermatozoa are very rare. The most extremecases involve finding <100 spermatozoa, and sometimeswell below this number. This video shows a method ofovercoming this problem, by cryostoring single humanspermatozoa (Cohen et al ., 1997). It involves placing themin a zona pellucida after its oocyte has been removed. Thesequences in the video include the initial preparation ofzonae pellucidae, which came from oocytes that wereimmature when aspirated from their follicles, eggs whichdisplayed abnormal fertilization, and unfertilized mouseand hamster oocytes. Fine lesions were made in the zonaeusing mechanical methods. The ooplasm was thenaspirated from the oocytes and eggs, to provide emptyzonae for storage. The example shows a hamster profile.Up to 15 spermatozoa, are then isolated and injected intothe evacuated zona pellucida, which was crypopreservedusing glycerol; this cryopreservation procedure is notshown on this video. The final sequences illustrate therecovery of the frozen/thawed spermatozoa. In practice,only one zona pellucida among 50 was not recovered aftercryopreservation, and no spermatozoa were lost throughthe narrow incisions in the zona pellucida. Fertilizationrates using the cryopreserved spermatozoa for ICSI wereabove 65%.AcknowledgementsThis video was prepared with the assistance of Mina Alika-ni, Adrienne Reing, Toni Ferrarra, Sasha Sadowy and TimSchimmel.References

Gamete intra-Fallopian transfer with cryopreserved donor semen following AID failure

Results are presented of gamete intra-Fallopian transfer with cryopreserved donor semen in cases of male infertility with azoospermia or extreme oligozoospermia. The female partners had no detectable infertility problems and they had first received 10 cycles of intracervical AID without success. Forty-one GIFT cycles performed on 22 such patients gave an overall pregnancy rate of 26.8% (11/41). In 64% (9/14) of these cases, pregnancy occurred after the first GIFT attempt. From these results it is suggested that GIFT with donor semen may be a reasonable alternative after AID failure. The probable reasons for success of GIFT are discussed.

Suppression of sperm function by depot medroxyprogesterone acetate and testosterone enanthate in steroid male contraception*†

AbstractTen normal men were given three monthly intramuscular injections of 200 mg of depot medroxyprogesterone acetate (MPA) and 250 mg of testosterone (T) enanthate. Six men became azoospermic, while four remained oligozoospermic, with a mean sperm density of 1.5 ± 0.3 standard error of the mean million/mi. Zona-free hamster oocyte penetration was abolished in all oligozoospermic samples at the end of treatment. Twenty of the 21 oligozoospermic samples yielding at least 0.6 to 5.0 million motile spermatozoa showed a complete absence of oocyte penetration. Semen parameters returned to normal, although some took up to 12 months. These findings demonstrated an antifertility action of MP A and T enanthate on the functional capacity of residual spermatozoa and support the view that extreme oligozoospermia may be a tenable target for reversible steroid male contraception.