DataSheet1_Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis.PDF

Introduction: Autologous transplantation of spermatogonial stem cells (SSCs) isolated from cryopreserved testicular biopsies obtained before oncological treatment could restore fertility in male childhood cancer survivors. There is a clear necessity for in vitro propagation of the limited SSCs from the testicular biopsy prior to transplantation due to limited numbers of spermatogonia in a cryopreserved testicular biopsy. Still, there is no consensus regarding their optimal culture method.Methods: We performed a systematic review and meta-analysis of studies reporting primary testicular cell cultures of human and non-human primate origin through use of Pubmed, EMBASE, and Web of Science core collection databases. Of 760 records, we included 42 articles for qualitative and quantitative analysis. To quantify in vitro spermatogonial propagation, spermatogonial colony doubling time (CDT) was calculated, which measures the increase in the number of spermatogonial colonies over time. A generalized linear mixed model analysis was used to assess the statistical effect of various culture conditions on CDT.Results: Our analysis indicates decreased CDTs, indicating faster spermatogonial propagation in cultures with a low culture temperature (32°C); with use of non-cellular matrices; use of StemPro-34 medium instead of DMEM; use of Knockout Serum Replacement; and when omitting additional growth factors in the culture medium.Discussion: The use of various methods and markers to detect the presence of spermatogonia within the reported cultures could result in detection bias, thereby potentially influencing comparability between studies. However, through use of CDT in the quantitative analysis this bias was reduced. Our results provide insight into critical culture conditions to further optimize human spermatogonial propagation in vitro, and effectively propagate and utilize these cells in a future fertility restoration therapy and restore hope of biological fatherhood for childhood cancer survivors.</p

HSROC curve.

<p>Hierarchical summary receiver operating characteristic (HSROC) plot of sperm DNA fragmentation for prediction of (clinical) pregnancy. Each circle on the plot represents the pair of sensitivity and specificity from a study and the size of the circle is scaled according to the sample size of the study. The solid red block represents the summary sensitivity and specificity, and this summary point is surrounded by a 95% confidence region (yellow dashed line) and 95% prediction region (green dotted line). Sperm DNA fragmentation in the prediction of (clinical) pregnancy for all studies and all cut-off values of the DNA fragmentation index reported: (A) SCSA, (B) SCD test, (C) TUNEL assay and (D) alkaline Comet assay. AUC: Area under the curve; HSROC: Hierarchical summary receiver operating characteristics.</p

Overall risk of bias in meta-analysis.

<p>This figure illustrates the overall risk of bias in the meta-analysis. The horizontal axis represents the number of studies included. The color of the bars represent the risk of bias. Yellow: high risk, blue: low risk and grey: unclear risk.</p

Flowdiagram of search and selection strategy in a systematic review and meta-analysis of sperm DNA fragmentation tests and pregnancy rates after MAR.

<p>Legend: not applicable.</p

Forest plot.

<p>Forest plot of sperm DNA fragmentation according to the DNA fragmentation index for predicting pregnancy. The plot shows study-specific estimates of sensitivity and specificity (with 95% confidence intervals). The studies are ordered according to the type of treatment: (A) SCSA, (B) SCD test, (C) TUNEL assay and (D) alkaline Comet assay. CI: confidence interval.</p

Descriptive data of all studies for the meta-analysis regarding SCSA, SCD test, TUNEL assay and Comet assay as tools for measure sperm DNA fragmentation.

<p>Descriptive data of all studies for the meta-analysis regarding SCSA, SCD test, TUNEL assay and Comet assay as tools for measure sperm DNA fragmentation.</p

List of studies excluded from the meta-analysis.

<p>List of studies excluded from the meta-analysis.</p

Study characteristics according to QUADAS II recommendations to report the risk of bias for patient selection and the concerns for applicability of data collected in manuscripts eligible for the meta-analysis.

<p>Study characteristics according to QUADAS II recommendations to report the risk of bias for patient selection and the concerns for applicability of data collected in manuscripts eligible for the meta-analysis.</p