Extend the Survival of Human Sperm In Vitro in Non-Freezing Conditions: Damage Mechanisms, Preservation Technologies, and Clinical Applications

Cheng, Qingyuan; Li, Liman; Jiang, Min; Liu, Bo; Xian, Yuezhong; Li, Shasha; Liu, Xiao; Zhao, Wenrui; Li, Fuping

doi:10.3390/cells11182845

Cited by 11 publications

(5 citation statements)

References 282 publications

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“…However, in this study of Ramasamy et al , if no sperm was retrieved after mincing, the tissue was kept overnight, and enzymatic digestion followed only the next day. This longer incubation time is potentially associated with increased extracellular oxidative stress and possible negative effects on the DNA and fertilizing capacity of the retrieved sperm ( Cheng et al , 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval

Vloeberghs,

De Munck,

Racca

et al. 2023

Human Reproduction Open

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STUDY QUESTION What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval rates for patients with non-obstructive azoospermia (NOA)? SUMMARY ANSWER In addition to mechanical mincing, enzymatic digestion increased sperm retrieval (SR) rates in testicular biopsies of NOA patients. WHAT IS KNOWN ALREADY Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, controversy still exists about whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, e.g. enzymatic digestion, in order to optimize SR rates. STUDY DESIGN, SIZE, DURATION This retrospective single-centre cohort study included all patients who underwent their first testicular sperm extraction (TESE) by open multiple-biopsy method between January 2004 and July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a diagnosis of NOA based on histology were included. The primary outcome was SR rate after mincing and/or enzymes. The secondary outcome was cumulative life birth (CLB) after ICSI with fresh TESE and subsequent ICSI cycles with frozen TESE. PARTICIPANTS/MATERIALS, SETTING, METHODS Multiple biopsies were obtained from the testis, unilaterally or bilaterally, on the day of oocyte retrieval. Upon mechanical mincing, biopsies were investigated for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using collagenase type IV. Multivariable regression analysis was performed to predict CLB per TESE by adjusting for the following confounding factors: male FSH level, female age and requirement of enzymatic digestion to find sperm. MAIN RESULTS AND THE ROLE OF CHANCE We included 118 patients, of whom 72 (61.0%) had successful SR eventually. Spermatozoa were retrieved after mechanical mincing for 28 patients (23.7%; 28/118) or after additional enzymatic digestion for another 44 patients (37.2%; 44/118). Thus, of the 90 patients requiring enzymatic digestion, sperm were retrieved for 44 (48.9%). Male characteristics were not different between patients with SR after mincing or enzymatic digestion, in regard to mean age (34.5 vs 34.5 y), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%), varicocele (3.6 vs 4.6%) or histological diagnosis (Sertoli-cell only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%). Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) achieved a life birth (LB) after the injection with fresh testicular sperm (and fresh or frozen embryo transfers). Of the remaining 49 patients without LB, 34 (69.4%) had supernumerary testicular sperm frozen. Of these 34 patients, 19 (55.9%) continued ICSI with frozen testicular sperm, and 9/19 (47.4%) had achieved a LB after ICSI with frozen testicular sperm. Thus, the total CLB was 32/118 (27.1%) per TESE or 32/72 (44.4%) per TESE with sperm retrieved. Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 y; p = 0,042) was significantly lower in the group with a live birth, compared to those without. The CLB with testicular sperm obtained after enzymatic digestion was 31.8% (14/44), while the CLB with sperm obtained after mincing alone 64.3% (18/28). Multivariable logistic regression analysis showed that when enzymatic digestion was required, it was associated with a significant decrease in CLB per TESE (OR: 0.23 [0.08-0.7]; p = 0.01). LIMITATIONS, REASONS FOR CAUTION Limitations of the study are related to the retrospective design. However, the selection of only patients with NOA, and specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE, strengthens our findings. WIDER IMPLICATIONS OF THE FINDINGS Enzymatic processing increases the SR rate from testicular biopsies of NOA patients compared to mechanical mincing only, demonstrating the importance of an appropriate laboratory protocol. However, NOA patients should be counselled that when sperm have been found after enzymatic digestion, their chances to father a genetically own child may be lower compared to those not requiring enzymatic digestion. STUDY FUNDING/COMPETING INTEREST(S) None reported TRIAL REGISTRATION NUMBER N/A

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Section: Discussionmentioning

confidence: 99%

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval

Vloeberghs,

De Munck,

Racca

et al. 2023

Human Reproduction Open

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“…Furthermore, our study revealed that during 8 h of storage, velocity parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) did not vary significantly between the two extenders, whereas after 24 h, the VCL, VAP, LIN and WOB decreased significantly only in SM extender. Normally, extenders play a crucial role in preserving sperm due to their richness in energy compounds, buffers and salts as well as antioxidants and antimicrobial substrates (Bustani & Baiee, 2021; Cheng et al, 2022). Hence our findings in terms of motility and velocity parameters could be probably linked to Duragen® composition supposed to contain efficient energy substrates compared to SM.…”

Section: Discussionmentioning

confidence: 99%

Comparative evaluation of Duragen® and skim milk extenders to enhance ram semen quality and fertility: A promising alternative in ovine artificial insemination

El Amiri,

Ben Moula,

El Khelfaoui

et al. 2023

Reprod Domestic Animals

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This study aimed to investigate the effectiveness of Duragen® and skimmed milk (SM) extenders on the quality parameters, bacterial load and fertilization ability of stored ram semen. A total of 50 ejaculates from Sardi rams (n = 5) aged 2.5–3 years, were collected and stored in Duragen® and SM at 15°C. The motilities and velocity parameters generated by the CASA system were then evaluated at 0, 8 and 24 h of storage. Afterward, bacterial loads of sperm extended in Duragen® and SM were determined at 0, 5 and 24 h of incubation. In addition, ewes (n = 100) aged 2 years, have been chosen in the same herd. The selected ewes were then synchronized and inseminated using semen extended in Duragen® and SM and stored for 5 h at 15°C. The results revealed that total and progressive motilities, straight velocity (VSL), straightness (SRT), lateral head displacement (ALH) and beat cross frequency (BCF) were not affected by the extender type after 24 h of storage (p > .05). However, curvilinear velocity (VCL), velocity average path (VAP), linearity (LIN) and wobble (WOB) showed higher values in Duragen® compared with SM extender after 24 h of storage (p < .05). Bacterial loads were observed mainly in sperm stored in SM at 5 h (183 UFC/mL) and at 24 h (357 UFC/mL) of incubation. However, the only case showing a bacterial load in Duragen® is when the storage time attains 24 h (199 UFC/mL). Concerning fertility, sperm diluted in both extenders resulting in high fertility rates which reaches 66% and 73% for Duragen® and SM, respectively, with no statistical difference (p > .05). In summary, Duragen® extender decreased bacterial load in stored semen and maintained high ram sperm quality and fertility. These findings suggest that Duragen® extender could be used as SM alternative in ovine artificial insemination (OAI).

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“…Yet, during sperm storage, the pH decreases gradually due to accumulation of acidic metabolites. It is generally accepted that acidic environments are toxic to spermatozoa, rendering them immobilized [ 70 , 71 ]. Since HCO 3 − acts as a natural buffering agent of semen, we may hypothesize that NBC overexpression in cryopreserved spermatozoa may only partially be associated with premature capacitation.…”

Section: Discussionmentioning

confidence: 99%

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A

Benko

Fialková²,

Žiarovská³

et al. 2022

IJMS

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Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

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Extend the Survival of Human Sperm In Vitro in Non-Freezing Conditions: Damage Mechanisms, Preservation Technologies, and Clinical Applications

Cited by 11 publications

References 282 publications

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval

Comparative evaluation of Duragen® and skim milk extenders to enhance ram semen quality and fertility: A promising alternative in ovine artificial insemination

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A

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