Association of X Chromosome Aberrations with Male Infertility

Male infertility is caused by spermatogenetic failure, clinically noted as oligoor azoospermia. Approximately 20% of infertile patients carry a genetic defect. The most frequent genetic defect leading to azoospermia (or severe oligozoospermia) is Klinefelter syndrome (47, XXY), which is numerical chromosomal abnormality and Y- structural chromosome aberration. The human X chromosome is the most stable of all human chromosomes. The X chromosome is loaded with regions of acquired, rapidly evolving genes. The X chromosome may actually play an essential role in male infertility and sperm production. Here we will describe X chromosome aberrations, which are associated with male infertility.

The mutation c.346-1G > a in SOHLH1 impairs sperm production in the homozygous but not in the heterozygous condition

Nonobstructive azoospermia (NOA) is an important cause of male infertility, and the genetic pathogenesis is still incompletely understood. The previous study reported that heterozygous mutation of c.346-1G > A in SOHLH1 was identified in two NOA patients and suggested it is the pathogenic factor for NOA. However, in our research, this heterozygous mutation was confirmed in three Chinese infertile patients who were suffered from teratozoospermia, but they had normal sperm number. Intriguingly, a homozygous mutation of c.346-1G > A in SOHLH1 was detected in a severe oligozoospermia (SOZ) patient, characterized with severely decreased sperm count. Notably, we unprecedently revealed this homozygous mutation of c.346-1G > A in SOHLH1 lead to the sharp decrease in various germ cells and spermatogenesis dysfunction, which is similar with the phenotype of Sohlh1 knockout male mice. Moreover, western blotting confirmed that the homozygous mutation declined SOHLH1 protein expression. Additionally, we correlated the good prognosis of intracytoplasmic sperm injection (ICSI) in the patients carrying the mutation of c.346-1G > A in SOHLH1. Thus, we suggested that the heterozygous mutation of c.346-1G > A in SOHLH1 is responsible for teratozoospermia, and this homozygous mutation in SOHLH1 impairs spermatogenesis and further leads to the reduced sperm count, eventually causing male infertility, which unveils a new recessive-inheritance pattern of SOHLH1-associated male infertility initially.

P–014 Effect of microsurgical varicocelectomy on fertility outcome and treatment plans of patients with severe oligozoospermia: An original report and meta-analysis

Study question Does microsurgical subinguinal varicocelectomy (MSV) improve semen parameters and fertility outcomes of patients with severe oligozoospermia (SO) and clinical varicocele? Summary answer MSV significantly improves semen parameters of patients with SO and can broaden their fertility treatment options. What is known already: Varicocele ligation has been proven to restore semen parameters and improve pregnancy rates in men with clinically palpable disease. However, its effect in men with SO is less clearly elucidated. Patients with SO are candidates for in vitro fertilization and intracytoplasmic sperm injection. Improvements in semen quality following varicocele ligation in this patient population may broaden their fertility options. While few studies indicate an improvement in semen parameters, reports revealing a negative outcome following surgery in this patient group were also published. Study design, size, duration This original report and meta-analysis examined the impact of MSV on semen parameters and fertility outcomes of men with SO. A retrospective chart review of 85 patients was conducted on patients with SO who underwent MSV. A literature search was carried out according to the PRISMA guidelines using the key words “severe oligozoospermia” and “varicocele”. 8 scientific articles (including the current study) reporting the impact of MSV on men with SO were included. Participants/materials, setting, methods Changes in semen parameters postoperatively were compared with pre-operative results. The reported natural pregnancy rates were also calculated. The Wilcoxon signed-rank test was used to compare semen and hormone values before and after varicocelectomy. The Chi-squared test was used to assess the changes in TMSC groups after surgery. The meta-analysis was performed using comprehensive meta-analysis software (Biostat, Englewood, NJ, USA). Statistical significance was set at α = 0.05. The random-effects model was used to adjust for heterogeneity. Main results and the role of chance The original study reported significant improvements in sperm concentration (p < 0.001), total motility (p = 0.003), progressive motility (p = 0.002) and TMSC (p < 0.001) was following the surgery. in semen parameters following surgery. 78 patients had a pre-operative TMSC < 5 million. Following surgery, 9 (11.5%) patients had a TMSC between 5–9 million, while 14 (17.9%) patients had a TMSC > 9 million. The meta-analysis shows a statistically significant increase in sperm count following surgery (MD 5.64, 95% CI, 4.195–7.090, p = 0.00) with an acceptable degree of heterogeneity (Q value= 8.75, p = 0.188, I²= 31.5%). Similarly, the total motility significantly increased by 7.77% (p = 0.001) following surgery (95% CI, 3.248–12.297), however, with considerable heterogeneity among the reported results (Q value= 34.4, p < 0.001). TMSC was assessed by three studies, including ours. The meta-analysis shows a significant increase in TMSC following surgery (MD 8.44 million sperm, 95% CI, 4.648–12.228, p < 0.001) (Q Value= 2.53, p = 283, I²=20.7%). A total of 6 studies reported the natural pregnancy rate of patients with SO who underwent surgery. Out of 530 patients with preoperative SO, 146 patients achieved natural pregnancy following surgery indicating that the reported pregnancy rate was 27.5%. Limitations, reasons for caution One limitation to the original study is its relatively small sample size. However, this was compensated by conducting a meta-analysis and reporting the outcome of 601 patients with SO who underwent varicocele ligation. Another limitation is the retrospective nature of the study design. Wider implications of the findings: 29.5% of SO patients in the original study became eligible for IUI following varicocelectomy. Meta-analysis showed that 27.5% of patients achieved natural conception following surgery. Such information is beneficial during patient counselling and needs to be measured against the financial and clinical implications in order to make sound treatment decisions. Trial registration number NA

P–020 AZFc partial microdeletions of the Y chromosome is associated with severe oligozoospermia among Iranian men

Study question Are AZFc partial deletions correlated with severe oligozoospermia in Iranian men? Can we consider them as risk factors for infertility? Summary answer The frequency of total partial AZFc microdeletions was significantly higher in the oligozoospermia group compared to control group (8% vs. 3%, P = 0.028). What is known already Among many factors affecting male infertility, the second most common genetic factor is Y chromosome microdeletion. Some studies on partial AZFc microdeletions (especially on three major types; gr/gr, b1/b3 and b2/b3) have associated them with impaired spermatogenesis (azoospermia and oligozoospermia) in infertile men from different ethnicities. This finding is attributed to differences in alterations in pattern of DAZ/CDY1 copy numbers as spermatogenesis related genes. Study design, size, duration 200 oligozoospermic (sperm count <5 mil./mL) and 200 fertile men were included as case and control groups, respectively. Individuals with karyotype abnormalities, complete microdeletions in AZF regions, infections, hypogonadism, history of chemotherapy and radiation, cryptorchidism or history of orchiopexy were not included. The study was approved by the Royan Institute Ethics Committee. Written informed consents were obtained from each participant. Participants/materials, setting, methods Total DNA from peripheral blood was used to amplify six sequence-tagged sites (STS) markers through multiplex PCR to detect AZFc partial deletions according to previous studies. Patterns of deletion in DAZ and CDY1 copies were determined through PCR- RFLP. Main results and the role of chance The frequency of AZFc partial microdeletions was 8% in oligozoospermic men (16/200) which was significantly higher compared to 3% in control group (6/200) (P = 0.028). Hence, partial deletions may be considered as a risk factor for the male infertility in Iranian population. Also, gr/gr showed a higher frequency in oligozoospermic group (4%) compared to controls (1.5%) (P = 0.126). The combination of DAZ1/2+CDY1b was the most observed deletion pattern in 8 oligozoospermic men with gr/gr deletion (75%), while among 3 controls with gr/gr, DAZ3/4+CDY1a (2 out of 3) and DAZ3/4+CDY1b (1 out of 3) were detected. Therefore, DAZ1/2+CDY1b can be correlated to oligozoospermia. Limitations, reasons for caution In order to achieve stronger statistical results, a larger sample size is of more help. Wider implications of the findings: Risk of vertical transmission to male offspring and expansion in the size of deletions should be considered when providing ART services to infertile men. Genetic counseling is suggested in oligozoospermic men. Trial registration number -

O-227 Pre-treatment semen parameters in haematological malignancies. An analysis of sequential samples

Study question Does Leukaemia affect spermatogenesis more adversely than Hodgkin’s lymphoma and is the effect consistent in sequential samples? Summary answer LLeukaemia is associated with a higher incidence of azoospermia, oligozoospermia and asthenozoospermia compared to Hodgkin’s lymphoma. These findings were consistent in sequential samples. What is known already Hodgkin’s lymphoma (HL) and leukaemias are common haematological malignancies that affect young men. Although not all treatments for these malignancies are gonadotoxic, there is evidence that malignancy affects sperm quality. Our own analysis in over 3000 men revealed that a diverse group of malignancies affected semen parameters adversely. There is concern that a single sample analysis may not reveal the true state due to varied period of abstinence and naturally occurring variation in semen quality. Leukaemia and lymphoma are systemic diseases; leukaemia usually runs a more torrid course whilst HL a more indolent course and therefore may variably affect spermatogenesis. Study design, size, duration A retrospective analysis was performed on 125 men with leukaemia and 303 men with HL. Only those men who had sequential semen analyses (1 and 2) within a month were included. Volume, sperm concentration and motility were the selected parameters in samples 1 and 2. Time period was April 1980 to January 2021. Participants/materials, setting, methods We included all post-pubertal men diagnosed with 2 most common haematological malignancies (Hodgkin’s lymphoma and leukaemia) in our database. Patient’s demographics, cancer diagnosis and semen parameters were extracted from a secure electronic database and analysed using MS Excel. Cancer diagnoses were obtained from referral letters from oncologists. Differences between samples 1 and 2 were tested using Fisher’s test, and odds ratios (OR) were calculated for the two malignancy groups. Main results and the role of chance We analysed 250 samples in 125 men with leukaemia and 606 samples in 303 men with HL. The mean intervals between the two semen samples were similar; 4.4 (1-30) and 3.8 (1-30) days. There were 95.7% of men <40 years in the HL group and 90.4% in the leukaemia group. There was no significant difference in the incidence of low volume (<1.5ml), sperm concentration or motility between samples 1 and 2 in both groups. Oligospermia was more frequently associated with leukaemia (OR 2.22, CI 95%, 1.44-3.43). Although the incidence of severe oligozoospermia was similar between the two cancer groups (OR 0.99, 95% CI 0.55 - 1.99), azoospermia was observed to have a greater association with leukaemia than HL (OR 3.22, 95% CI 1.57-6.63). There was also a greater association of asthenozoospermia with leukaemia compared to HL (OR 2.76, 95% CI 1.76-4.35). As there was consistency between samples 1 and 2 in both groups, odds ratio calculation for sample 2 revealed similar results as for sample 1. Limitations, reasons for caution As we selected men with at least two semen samples on two separate occasions, we had to exclude men with single samples which substantially reduced the number of participants. Types of leukaemia and the stage of disease in HL were not analysed. Wider implications of the findings Our findings are pertinent when counselling men about fertility preservation even in the absence of planned gonadotoxic treatment. Awareness about increased azoospermia incidence may help plan oncoTESE procedures. Our findings could form a basis for studies examining spermatogenesis pathways in haematological malignancies. Trial registration number not applicable

The genetic causes of infertility in patients with oligozoospermia and azoospermia in Turkish population

Objective: Advances in the science of genetics and the development of assisted reproductive techniques focus on the genetic causes of infertility. The aim of this research is to reveal genetic abnormalities in terms of sex chromosome aneuploidy and Y chromosome microdeletions. Material and Methods: A total of 350 patients with azoospermia or severe oligozoospermia were selected. After general examination of the patients and laboratory investigations were performed, cartoypes and Y chromosome microdeletions were examined. Results: A total of 225 infertile men with non-obstructive azoospermia (NOA) and 125 infertile men with oligozoospermia were enrolled into the study. The overall cytogenetic anomaly rate was 16%. Chromosomal changes were detected in 32 of 350 (9.1%) cases. The most common genetic anomaly was 47, XXY (Klinefelter syndrome) and the incidence was 11.5% in NOA group. This rate was 3.2% in oligozoospermia group. Y chromosome microdeletions were detected in 24 (6.8%) patients and similarly, it was observed more frequently in the NOA group than in the oligozoospermia group. Conclusion: The incidence of genetic causes have been increasing with the severity of infertility. As a result, genetic screening and appropriate genetic counseling are needed before the use of assisted reproductive techniques. Keywords: azospermia, chromosome, infertility, microdeletion, oligozoospermiaage

Cryopreservation of sperm before gonadotoxic treatment at the University Hospital Brno in the years 1995–2020

Summary Objective: Sperm cryopreservation before gonadotoxic treatment is the basic and mos teffective method of preserving reproduction, which can be used during adolescence. The communication summarizes 26 years of experience in the operation of an oncological sperm bank, analyzes spermiograms of oncological patients, assesses the relationship between sperm pathology and diagnosis, and determines the number of deaths and the use of frozen sperm. Methods: During the existence of CAR 01 (assisted reproduction center), more than 50,000 spermiograms were performed. From January 1995 to December 2020, a total of 24,729 men were examined within the sperm bank, of which 1,448 (5.9%) had an oncological diagnosis. The spermiograms were evaluated according to current WHO (World Health Organization) manuals. Cryopreservation of sperm has undergone a major development. The rules for the storage of frozen cells have been laid down by Act No. 296/2008 Coll. since 2008. In 2019, the methodology „Cryopreservation of reproductive cells and tissues in patients before cancer treatment“ was updated. In all cases, the standard thawing technique was used. The sperms were processed by the swim-up method. As part of the treatment with assisted reproduction methods, oocytes were fertilized by the ICSI (intracytoplasmatic sperm injection) micromanipulation technique. Results: Out of 1,448 examined spermiograms in men with oncological diagnoses, testicular cancer was present in 43.7% of patients and malignant diseases of lymphatic and hematopoietic tissue were found in 24.1%, of which 70,1% included Hodgkin‘s lymphomas and 29,9% were non-Hodgkin‘s lymphomas. Leukemia was found in 7.9%, bone and cartilage cancers in 6.8%. The age of the clients of the whole group ranged from 13 to 64 years (27.2 ± 6.8 years). A total of 38.3% of men had normozoospermia, 54.2% of spermiograms showed pathological findings in 1 to 3 evaluated parameters and 7.5% of patients had azoospermia. Severe asthenozoospermia (mobility ≤ 10%) was detected in 57.2% of men and severe oligozoospermia (concentration ≤ 1 × 106 mm3) in 22.3% of patients. The lowest values of the spermiogram were found in men with testicular cancer; the best values were seen in CNS (central nervous system) cancers. The cryopreservation of sperm was performed in 1,340 cases (92.5%). So far, a total of 160 men (11.9%) have used frozen sperm, of which 6.2% in our center. In these 83 cases, the ICSI technique was always used, 38 clinical pregnancies (45.8%) and 32 births were achieved. We have registered 424 completed storages of semen (31.6%), of which 148 (11.0% of all oncology patients) were made due to death and the others at patients’ request. Using the sperm of the dead is a specific issue. Conclusion: In cancer patients, sperm pathologies occur in high percentage. The lowest spermiogram values were found in men with testicular cancer. It is necessary to take into account long-term storage and fertilization by micromanipulation methods. The number of men who die is significantly higher than the number of those who use sperm to treat infertility. Cryopreservation of sperm should be offered to each patient prior to the therapy leading to the destruction of spermatogenesis.