What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

Research question From a clinical perspective, which parameters grant optimal cryopreservation of mouse testicular cell suspensions? Design We studied the effect of different cryopreservation rates, the addition of sugars, different vessels and the addition of an apoptotic inhibitor on the efficiency of testicular cell suspension cryopreservation. After thawing and warming, testicular cell suspensions were transplanted to recipient mice for further functional assay. After selecting the optimal cryopreservation procedure, a second experiment compared the transplantation efficiency between the selected freezing protocol and fresh testicular cell suspensions. Results Multiple- and single-step freezing did not differ significantly in terms of recovered viable cells (RVC) (33 ± 28% and 38 ± 25%). The addition of sucrose did not result in a higher RVC (33 ± 20%). Cells frozen in vials recovered better than those frozen in straws (52 ± 20% versus 33 ± 20%; P = 0.0049). The inclusion of an apoptosis inhibitor (z-VAD[Oe]-FMK) significantly increased the RVC after thawing (61 ± 18% versus 50 ± 17%; P = 0.0480). When comparing the optimal cryopreservation procedure with fresh testicular cell suspensions, a lower RVC (63 ± 11% versus 92 ± 4%; P < 0.0001) and number of donor-derived spermatogonial stem cell colonies per testis (34.04 ± 2.34 versus 16.78 ± 7.76; P = 0.0051) were observed. Conclusion Upon freeze–thawing or vitrification–warming, and assessment of donor-derived spermatogenesis after transplantation, Dulbecco's modified Eagle's medium supplemented with 1.5M dimethyl-sulphoxide, 10% fetal calf serum and 60 µM of Z-VAD-(OMe)-FMK in vials at a freezing rate of −1°C/min was optimal

Testis weight.

<p>Bodyweight (A) and testicular weight (B) of the adult treated rats at the time of euthanasia. Bodyweight (C) and testicular weight (D) of the prepubertal treated rats at the time of euthanasia. Variables with a different letter are statistically different. If no letters are indicated, no statistical differences were found in the graph. FC: fertile controls, O: rats treated with unilateral orchiectomy, B: rats treated with busulfan, O+B: rats treated with busulfan and unilateral orchiectomy.</p

Testosterone levels.

<p>Evolution of testosterone levels of rats treated at adult (a) or prepubertal (b) age. Variables with a different letter are statistically different. FC: fertile controls, O: rats treated with unilateral orchiectomy, B: rats treated with busulfan, O+B: rats treated with busulfan and unilateral orchiectomy, D: day.</p

Evaluation of spermatogenesis and number of spermatogonia.

<p>a) A UCHL1 staining was performed to evaluate the number of spermatogonia (white arrow) per tubule. All pictures show testis sections of prepubertal treated rats. Left row: DAPI, middle row: UCHL1, right row: merge UCHL1+DAPI. b) A HE staining was performed to evaluate spermatogenesis. All pictures show testis sections of prepubertal treated rats. Thirty percent of the rats that received the combined treatment at prepubertal age showed SCO tubules. FC: fertile controls, O: rats treated with unilateral orchiectomy, B: rats treated with busulfan, O+B: rats treated with busulfan and unilateral orchiectomy.</p

Mating.

<p>Number of litters produced during the whole mating experiment per adult (a) or prepubertal (c) treated rat per gestation period during 4 months of mating. Mean number of pups per litter produced per adult (b) or prepubertal (d) treated rat per gestation period. Variables with a different letter are statistically different. If no letters are indicated, no statistical differences were found in the graph. FC: fertile controls, O: rats treated with unilateral orchiectomy, B: rats treated with busulfan, O+B: rats treated with busulfan and unilateral orchiectomy.</p

The Effects of Heatwaves on Human Morbidity in Primary Care Settings: A Case-Crossover Study

Purpose: This study assesses the potential acute effects of heatwaves on human morbidities in primary care settings. Methods: We performed a time-stratified case-crossover study to assess the acute effects of heatwaves on selected morbidities in primary care settings in Flanders, Belgium, between 2000 and 2015. We used conditional logistic regression models. We assessed the effect of heatwaves on the day of the event (lag 0) and X days earlier (lags 1 to X). The associations are presented as Incidence Density Ratios (IDR). Results: We included 22,344 events. Heatwaves are associated with increased heat-related morbidities such as heat stroke IDR 3.93 [2.94&ndash;5.26] at lag 0, dehydration IDR 3.93 [2.94&ndash;5.26] at lag 1, and orthostatic hypotension IDR 2.06 [1.37&ndash;3.10] at lag 1. For cardiovascular morbidities studied, there was only an increased risk of stroke at lag 3 IDR 1.45 [1.04&ndash;2.03]. There is no significant association with myocardial ischemia/infarction or arrhythmia. Heatwaves are associated with decreased respiratory infection risk. The IDR for upper respiratory infections is 0.82 [0.78&ndash;0.87] lag 1 and lower respiratory infections (LRI) is 0.82 [0.74&ndash;0.91] at lag 1. There was no significant effect modification by age or premorbid chronic disease (diabetes, hypertesnsion). Conclusion: Heatwaves are associated with increased heat-related morbidities and decreased respiratory infection risk. The study of heatwaves&rsquo; effects in primary care settings helps evaluate the impact of heatwaves on the general population. Primary care settings might be not suitable to study acute life-threatening morbidities

The effects of heatwaves on human morbidity in primary care settings : a case-crossover study

Purpose: This study assesses the potential acute effects of heatwaves on human morbidities in primary care settings. Methods: We performed a time-stratified case-crossover study to assess the acute effects of heatwaves on selected morbidities in primary care settings in Flanders, Belgium, between 2000 and 2015. We used conditional logistic regression models. We assessed the effect of heatwaves on the day of the event (lag 0) and X days earlier (lags 1 to X). The associations are presented as Incidence Density Ratios (IDR). Results: We included 22,344 events. Heatwaves are associated with increased heat-related morbidities such as heat stroke IDR 3.93 [2.94–5.26] at lag 0, dehydration IDR 3.93 [2.94–5.26] at lag 1, and orthostatic hypotension IDR 2.06 [1.37–3.10] at lag 1. For cardiovascular morbidities studied, there was only an increased risk of stroke at lag 3 IDR 1.45 [1.04–2.03]. There is no significant association with myocardial ischemia/infarction or arrhythmia. Heatwaves are associated with decreased respiratory infection risk. The IDR for upper respiratory infections is 0.82 [0.78–0.87] lag 1 and lower respiratory infections (LRI) is 0.82 [0.74–0.91] at lag 1. There was no significant effect modification by age or premorbid chronic disease (diabetes, hypertesnsion). Conclusion: Heatwaves are associated with increased heat-related morbidities and decreased respiratory infection risk. The study of heatwaves’ effects in primary care settings helps evaluate the impact of heatwaves on the general population. Primary care settings might be not suitable to study acute life-threatening morbidities