Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30  ×  10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

A simple method for repeated in vivo sperm collection from laboratory mice.

PURPOSE: Mouse spermatozoa for archiving laboratory mice or for in vitro fertilization (IVF) are routinely obtained from the cauda epididymis of adult males sacrificed for this purpose. To avoid the death of the donor, we tested whether a precisely timed interruption of the mating act could be used for repeated sperm collection from laboratory mice. METHODS: Sperm donors (B6D2F1) were mated with a receptive female, and mating behavior was observed. The stud was separated from the female 1-2 s after the onset of the ejaculatory shudder. The ejected copulatory plug with the yellowish viscous ejaculate was carefully removed from the penile cup. RESULTS: A total of 80 ejaculates were successfully obtained from 100 ejaculations. The latency to first mount was 1.1 ± 1.1 min (mean ± SD) and to ejaculation 8.1 ± 4.7 min. The average number of mounts to ejaculation was 10.5 ± 5.8, and the mean number of spermatozoa per collected ejaculate was 1.86 ± 1.05 × 106. An average fertilization rate of 76% was observed after IVF. CONCLUSIONS: Separating the stud from the female just before ejaculation is feasible, easy to learn, and requires no special equipment. The sperm count of collected ejaculates is lower than natural ejaculations, but higher than previous in vivo sperm collection methods achieved. We recommend this simple sperm collection method in mice, especially when the donor cannot be sacrificed and/or repeated sperm collection from the same animal is required for experimental purposes.

Practical problems in the posthumous retrieval of sperm.

This communication discusses the practical problems that arise during the collection and processing of sperm that have been retrieved posthumously. It is based on a small group, namely the last six men from whom we carried out posthumous retrieval. The reason for each retrieval, the method of that retrieval, the assessment of the sperm retrieved, the subsequent viability of the sperm and their storage method are discussed. The many ethical and legal problems that arise both before and after posthumous sperm retrieval are huge in their complexity. Therefore, they will not be discussed here and this communication will be limited to the practical aspects of posthumous sperm retrieval. The purpose of this communication is to make some suggestions that will facilitate such collections. The whole subject of posthumous sperm collection is gaining increasing clinical importance and has begun to interest the media as demonstrated by the recent national coverage in an Australian newspaper.

Sperm collection and characteristics analysis of the critically endangered Chinese pangolin (Manis pentadactyla).

The Chinese pangolin (Manis pentadactyla) is a critically endangered species. However, there is a paucity of research on the male reproductive gamete biology of this species. The present study was the first to systematically analyse the sperm characterization of the Chinese pangolin, including semen collection, sperm morphometry and ultrastructure. The semen of five male Chinese pangolins was successfully collected using the electroejaculation method. CASA (computer-assisted sperm analysis) was used to assess semen quality and take images for sperm morphometric analysis. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used for sperm ultrastructure observation. The results showed that the semen of the Chinese pangolin was yellow to pale yellow in colour, viscous, with a fishy odour, and a slightly alkaline pH of between 7.7 and 7.9. The head defects were the main sperm defects; there were 13 kinds of head defects counted in this study. The total sperm length, head length, head width and tail length were 67.62 ± 0.21 µm, 10.47 ± 0.06 µm, 1.33 ± 0.006 µm and 57.16 ± 0.20 µm, respectively. SEM observed that the spermatozoa had a rod-shaped head with a distinct apical ridge, which was different from most mammals and similar to that in avians and reptiles. Interestingly, TEM found that the acrosome membrane of the Chinese pangolin had a double membrane structure rather than a multiple bi-lamellar membrane structure as reported by the previous study. Collectively, this study contributes to the development of artificial breeding efforts and assisted reproductive techniques for the Chinese pangolin, as well as providing technical support for research on germplasm conservation of this species.

Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes).

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks' Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

The impact of percutaneous epididymal sperm aspiration on sperm quality in mice.

Abstract: In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits. Lay summary: In mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

Artificial Insemination in Queens in the Clinical Practice Setting: Protocols and challenges.

PRACTICAL RELEVANCE: Despite substantial advances in assisted reproductive techniques having been recently reported in cats, the use of these is limited and routine application is still far from being a reality in veterinary clinics. Nevertheless, there is an increasing demand from domestic cat breeders for artificial insemination (AI) techniques that are already commonly used in dogs. Where natural breeding is not possible in tom cats and queens of high breeding value, AI could offer a solution. Clinical challenges: AI in cats is more difficult than in other species - both in terms of semen collection/handling and oestrous cycle management given that ovulation must be induced. AIM: For practitioners wishing to perform AI in queens, there are challenges to overcome, and a good understanding of the techniques and procedures involved is pivotal. This review aims to contribute to improved knowledge by providing an overview of AI protocols, encompassing choice of breeding animals, procedures for semen collection, oestrus and ovulation induction, AI techniques and equipment. EQUIPMENT AND TECHNICAL SKILLS: Depending on the animals involved and the specific AI technique chosen, essential equipment may include an artificial vagina, electroejaculator, endoscope (sialendoscope, which can be fairly expensive) and special catheters for transcervical insemination. Other instrumentation and materials needed are typically readily available in a veterinary clinic. In general, no particular skills are needed to perform the procedures described in this review, with the exception of endoscopic transcervical catheterisation, where the ability to use an endoscope is required. EVIDENCE BASE: The information and advice/recommendations provided are based on specific feline research and reviews published in scientific peer-reviewed journals, animal reproduction textbooks, and presentations at national and international congresses. The authors also drew on their own clinical experience with regard to the choice of protocols and procedures presented in this review.

The Effect of the Time Interval From Sperm Processing to Intrauterine Insemination on the Pregnancy Outcomes of Infertile Women.

Objective: Intrauterine insemination (IUI) is the first-line treatment in couples suffering from various causes of subfertility and infertility. Considering the relatively low rate of pregnancy achieved with each cycle in this method, optimizing various steps in the process including the time interval from sperm collection to IUI may result in an increased rate of success. The goal of this study was to assess the impact of time intervals from the end of sperm processing to IUI (SP-IUI) on the pregnancy rate in IUI. Materials and methods: This single-center prospective cohort study evaluated couples with normal male partner sperm analysis and idiopathic female infertility undergoing IUI from 2018 to 2021. Cycles were stimulated using subcutaneous recombinant FSH and oral Letrozole. Ovulation was triggered using GnRH antagonist when the leading follicle's size reached greater than 14mm. The participants were placed in one of the three groups based on SP-IUI: group 1 (0-60 min), group II (60-90 min), and Group III: (>90 min). Results: 269 couples were included in the study. Sperm processing expectedly resulted in an increased concentration of total sperm count and sperm motility (P<0.001). The rate of chemical or clinical pregnancy, abortion, IUFD, multigestation, pregnancy, term birth, and ectopic pregnancy was not significantly different across study groups (P>0.05). Conclusion: The results of this study suggest that SP-IUI intervals evaluated in this study do not vary in terms of pregnancy rate or adverse pregnancy outcomes in IUI with normal male partner semen analysis. Hence, infertile couples can be flexible in the collection of semen specimens without time and site (at home or hospital) limitations.

Fertility preservation in young men with Klinefelter syndrome: A systematic review.

BACKGROUND: Klinefelter syndrome (KS) is the most common cause of genetic male infertility, as most patients present azoospermia. In the testis, a massive decrease in the number of germinal cells is observed and this can begin early in childhood. Thus, it is possible to collect spermatozoa after sperm collection or thanks to testicular sperm extraction (TESE), but the chances finding spermatozoa are decreasing with the age. Sperm collection or TESE should be performed as early as possible. When KS is diagnosed during childhood or teens, fertility preservation could be beneficial. The minimal age for proposing fertility preservation remains controversial and there is no current recommendation about fertility preservation in young men with KS. DESIGN: In this context, we have conducted a systematic review of the results of fertility preservation in young patients with KS to discuss the optimal age range for offering fertility preservation, including or not a TESE. RESULTS: Six articles were included in the systematic review, with patients between 13 and 24 years-old. Except for one, all young men agreed for sperm collection following masturbation. Azoospermia was diagnosed in all patients presenting homogenous KS. One study reported the presence of spermatozoa in the ejaculate of a young man with mosaic KS. Fifty-eight young man for whom ejaculated sperm collection was unsuccessful have benefited from TESE. Testicular spermatozoa were found and frozen in 27 patients out of the 58 (46.5%). The chances of freezing viable testicular sperm between 14 and 23 years of age do not appear to depend on age. CONCLUSION: Fertility preservation should be proposed in young men, but the optimal age for proposing the first sperm collection could be adapted according to the medical context and the psychological maturity of the young man.

Selection Criteria of Zebrafish Male Donors for Sperm Cryopreservation.

Selection criteria for sperm cryopreservation are highly relevant in zebrafish since sperm quality is particularly variable in this species. Successful cryopreservation depends on high-quality sperm, which can only be ensured by the selection of breeders. Consequently, male selection and management are a priority to improve cryopreservation, and therefore, this study aimed to characterize optimal age and sperm collection frequency in zebrafish. For this purpose, males from wild type (AB) and from a transgenic line [Tg(runx2:eGFP)] were sampled at 6, 8, 12, and 14 months. For each age, sperm were collected at time 0 followed by samplings at 2, 7, and 14 days of rest. Sperm quality was assessed according to motility and membrane viability parameters. Quality assessment showed that Tg(runx2:eGFP) displayed significantly higher motility than AB and younger males showed higher motility in both lines. Sperm collection frequency affected membrane viability. While AB fish recovered sperm viability after 14 days of rest, Tg(runx2:eGFP) could not recover. Consequently, it may be important to study the sperm quality of each zebrafish line before sperm cryopreservation. Taking into consideration the results achieved in both lines, sperm collection should be performed between 6 and 8 months of age with a minimum collection interval of 14 days.

Assisted Reproduction in the Male Cat.

The demand for feline semen collection, evaluation, and subsequent use is growing as a way to preserve important genetic materials. This article describes semen collection methods using a variety of techniques that can be applicable in almost any setting. Also discussed are cryopreservation methods that optimize sperm survival.

Coleta de esperma infértil em laboratório: prática médica ou sexual? / Infertile sperm collection in hospital laboratories: sexual or medical practice?

Os protocolos de assistência médica à procriação comportam um exame biológico do esperma, realizado graças à masturbação praticada em locais inadequados para uma prática geralmente considerada erótica. Realizou-se investigação etnográfica em dois grandes hospitais parisienses. Os homens e as mulheres que participaram manifestaram reações subjetivas, associando constrangimento, vergonha, desagrado e nojo diante da prática da masturbação nesse contexto. A confusão entre os registros médico e sexual, as dificuldades para des-sexualizar uma prática que permanece como tabu e a natureza estéril do esperma produzido nessas circunstâncias seriam a causa das reações emocionais experimentadas em relação ao esperma, quando este é produzido no contexto médico.

The protocols of medical assistance to procreation involve a biological examination of sperm, conducted through masturbation practiced in inappropriate places for a practice generally considered erotic. We conducted ethnographic research in two major Parisian hospitals. The men and women who participated expressed subjective reactions, involving embarrassment, shame, disgust and disgust on the practice of masturbation in this context. The confusion between the medical and sexual records, difficulty in de-sexualize a practice that remains taboo, and the sterile nature of sperm produced in such circumstances would be the cause of emotional reactions experienced in relation to sperm when it is produced in the medical context.