BACKGROUNDSubfertility affects approximately 15% of all couples, and a severe male factor is identified in 17% of these couples. While the etiology of a severe male factor remains largely unknown, prior gonadotoxic treatment and genomic aberrations have been associated with this type of subfertility. Couples with a severe male factor can resort to ICSI, with either ejaculated spermatozoa (in case of oligozoospermia) or surgically retrieved testicular spermatozoa (in case of azoospermia) to generate their own biological children. Currently there is no direct treatment for azoospermia or oligozoospermia. Spermatogonial stem cell (SSC) autotransplantation (SSCT) is a promising novel clinical application currently under development to restore fertility in sterile childhood cancer survivors. Meanwhile, recent advances in genomic editing, especially the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system, are likely to enable genomic rectification of human SSCs in the near future.OBJECTIVE AND RATIONALEThe objective of this review is to provide insights into the prospects of the potential clinical application of SSCT with or without genomic editing to cure spermatogenic failure and to prevent transmission of genetic diseases.SEARCH METHODSWe performed a narrative review using the literature available on PubMed not restricted to any publishing year on topics of subfertility, fertility treatments, (molecular regulation of) spermatogenesis and SSCT, inherited (genetic) disorders, prenatal screening methods, genomic editing and germline editing. For germline editing, we focussed on the novel CRISPR-Cas9 system. We included papers written in English only.OUTCOMESCurrent techniques allow propagation of human SSCs in vitro, which is indispensable to successful transplantation. This technique is currently being developed in a preclinical setting for childhood cancer survivors who have stored a testis biopsy prior to cancer treatment. Similarly, SSCT could be used to restore fertility in sterile adult cancer survivors. In vitro propagation of SSCs might also be employed to enhance spermatogenesis in oligozoospermic men and in azoospermic men who still have functional SSCs albeit in insufficient numbers. The combination of SSCT with genomic editing techniques could potentially rectify defects in spermatogenesis caused by genomic mutations or, more broadly, prevent transmission of genomic diseases to the offspring. In spite of the promising prospects, SSCT and germline genomic editing are not yet clinically applicable and both techniques require optimization at various levels.WIDER IMPLICATIONSSSCT with or without genomic editing could potentially be used to restore fertility in cancer survivors to treat couples with a severe male factor and to prevent the paternal transmission of diseases. This will potentially allow these couples to have their own biological children. Technical development is progressing rapidly, and ethical reflection and societal debate on the use of SSCT with ...

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Current cancer treatment regimens do not only target tumor cells, but can also have devastating effects on the spermatogonial stem cell pool, resulting in a lack of functional gametes and hence sterility. In adult men, fertility can be preserved prior to cancer treatment by cryopreservation of ejaculated or surgically retrieved spermatozoa, but this is not an option for prepubertal boys since spermatogenesis does not commence until puberty. Cryopreservation of a testicular biopsy taken before initiation of cancer treatment, followed by in vitro propagation of spermatogonial stem cells and subsequent autotransplantation of these stem cells after cancer treatment, has been suggested as a way to preserve and restore fertility in childhood cancer survivors. This strategy, known as spermatogonial stem cell transplantation, has been successful in mice and other model systems, but has not yet been applied in humans. Although recent progress has brought clinical application of spermatogonial stem cell autotransplantation in closer range, there are still a number of important issues to address. In this paper, we describe the state of the art of spermatogonial stem cell transplantation and outline the hurdles that need to be overcome before clinical implementation.

Background There is currently no concise systematic review or meta‐analysis addressing cardio‐metabolic risk factors in women experiencing infertility. Objectives To determine whether infertile women have higher levels of cardiovascular risk factors compared with fertile women. Search strategy We performed a systematic literature search using PubMed, Embase and CINAHL, Scopus and additional manual and bibliographic searches for relevant articles (end search date 6 November 2019). Selection criteria We selected studies that compared cardio‐metabolic risk factors in fertile and infertile women of reproductive age. Data collection and analysis At least two authors independently screened potentially eligible studies. Main results There was an increased presence of several cardio‐metabolic risk factors in infertile women compared with fertile women. Infertile women had statistically significant higher body mass index (BMI), increased total cholesterol (TC), low‐density lipoprotein cholesterol (LDL‐C) and triglycerides (TG) compared with fertile women. Fasting glucose, fasting insulin, homeostatic model assessment of insulin resistance (HOMA‐IR) and mean arterial pressure were not found to be different between fertile and infertile women. A subgroup analysis revealed that TC, fasting glucose and fasting insulin were increased, and high‐density lipoprotein was decreased only in women with polycystic ovarian syndrome compared with fertile women, whereas BMI, TG and LDL‐C were statistically significantly increased in women with any indication of infertility compared with fertile women. Conclusions Infertile women have a higher level of cardio‐metabolic risk factors compared with fertile women. This finding has clinical implications for infertile women in general, and those attempting to conceive through medically assisted reproduction. Tweetable abstract Infertile women appear to have a higher level of cardio‐metabolic risk factors compared with fertile women.

STUDY QUESTION Can transgender women cryopreserve germ cells obtained from their orchiectomy specimen for fertility preservation, after having used puberty suppression and/or hormonal treatment? SUMMARY ANSWER In the vast majority of transgender women, there were still immature germ cells present in the orchiectomy specimen, and in 4.7% of transgender women—who all initiated medical treatment in Tanner stage 4 or higher—mature spermatozoa were found, which would enable cryopreservation of spermatozoa or testicular tissue after having used puberty suppression and/or hormonal treatment. WHAT IS KNOWN ALREADY Gender affirming treatment (i.e. puberty suppression, hormonal treatment, and subsequent orchiectomy) impairs reproductive function in transgender women. Although semen cryopreservation is generally offered during the transition process, this option is not feasible for all transgender women (e.g. due to incomplete spermatogenesis when initiating treatment in early puberty, in case of inability to masturbate, or when temporary cessation of hormonal treatment is too disruptive). Harvesting mature spermatozoa, or testicular tissue harboring immature germ cells, from orchiectomy specimens obtained during genital gender-affirming surgery (gGAS) might give this group a chance of having biological children later in life. Previous studies on spermatogenesis in orchiectomy specimens showed conflicting results, ranging from complete absence of germ cells to full spermatogenesis, and did not involve transgender women who initiated medical treatment in early- or late puberty. STUDY DESIGN, SIZE, DURATION Histological and immunohistochemical analyses were performed on orchiectomy specimens from 214 transgender women who underwent gGAS between 2006 and 2018. Six subgroups were identified, depending on pubertal stage at initiation of medical treatment (Tanner stage 2-3, Tanner stage 4-5, adult), and whether hormonal treatment was continued or temporarily stopped prior to gGAS in each of these groups. PARTICIPANTS/MATERIALS, SETTING, METHODS All transgender women used a combination of estrogens and testosterone suppressing therapy. Orchiectomy specimen sections were stained with Mayer’s hematoxylin and eosin and histologically analyzed to assess the Johnsen score and the ratio of most advanced germ cell types in at least 50 seminiferous tubular cross-sections. Subsequently, immunohistochemistry was used to validate these findings using spermatogonia, spermatocytes or spermatids markers (MAGE-A3/A4, γH2AX, Acrosin, respectively). Possibilities for fertility preservation were defined as: preservation of spermatozoa, preservation of spermatogonial stem cells or no possibilities (in case no germ cells were found). Outcomes were compared between subgroups and logistic regression analyses were used to assess the association between the duration of hormonal treatment and the possibilities for fertility preservation. MAIN RESULTS AND THE ROLE OF CHANCE Mature spermatozoa were encountered in 4.7% of orchiectomy specimens, all from transgender women who had initiated medical treatment in Tanner stage 4 or higher. In 88.3% of the study sample orchiectomy specimens only contained immature germ cells (round spermatids, spermatocytes or spermatogonia, as most advanced germ cell type). In 7.0%, a complete absence of germ cells was observed, all these samples were from transgender women who had initiated medical treatment in adulthood. Cessation of hormonal treatment prior to gGAS did not affect the presence of germ cells or their maturation stage, nor was there an effect of the duration of hormonal treatment prior to gGAS. LIMITATIONS, REASONS FOR CAUTION Since data on serum hormone levels on the day of gGAS were not available, we were unable to verify if the transgender women who were asked to temporarily stop hormonal treatment 4 weeks prior to surgery actually did so, and if people with full spermatogenesis were compliant to treatment. WIDER IMPLICATIONS OF THE FINDINGS There may still be options for fertility preservation in orchiectomy specimens obtained during gGAS since a small percentage of transgender women had full spermatogenesis, which could enable cryopreservation of mature spermatozoa via a testicular sperm extraction procedure. Furthermore, the vast majority still had immature germ cells, which could enable cryopreservation of testicular tissue harboring spermatogonial stem cells. If maturation techniques like in vitro spermatogenesis become available in the future, harvesting germ cells from orchiectomy specimens might be a promising option for those who are otherwise unable to have biological children. STUDY FUNDING/COMPETING INTEREST None. TRIAL REGISTRATION NUMBER N/A.

STUDY QUESTIONIs testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice?SUMMARY ANSWERCancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls.WHAT IS KNOWN ALREADYSpermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically.STUDY DESIGN, SIZE, DURATIONThis study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months.PARTICIPANTS/MATERIALS, SETTING, METHODSNeonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497–517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR.MAIN RESULTS AND THE ROLE OF CHANCEWe found 9% (95% CI: 2–25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14–45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1–1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076).LARGE...

Spermatogenesis, starting with spermatogonial differentiation, is characterized by ongoing and dramatic alterations in composition and function of chromatin. Failure to maintain proper chromatin dynamics during spermatogenesis may lead to mutations, chromosomal aberrations or aneuploidies. When transmitted to the offspring, these can cause infertility or congenital malformations. The structural maintenance of chromosomes (SMC) 5/6 protein complex has recently been described to function in chromatin modeling and genomic integrity maintenance during spermatogonial differentiation and meiosis. Among the subunits of the SMC5/6 complex, non-SMC element 2 (NSMCE2) is an important small ubiquitin-related modifier (SUMO) ligase. NSMCE2 has been reported to be essential for mouse development, prevention of cancer and aging in adult mice and topological stress relief in human somatic cells. By using cultured primary mouse spermatogonial stem cells (SSCs), referred to as male germline stem (GS) cells, we investigated the function of NSMCE2 during spermatogonial proliferation and differentiation. We first optimized a protocol to generate genetically modified GS cell lines using CRISPR-Cas9 and generated an GS cell line. Using this GS cell line, we found that NSMCE2 was dispensable for proliferation, differentiation and topological stress relief in mouse GS cells. Moreover, RNA sequencing analysis demonstrated that the transcriptome was only minimally affected by the absence of NSMCE2. Only differential expression of appeared highly significant, but with SGSM1 protein levels being unaffected without NSMCE2. Hence, despite the essential roles of NSMCE2 in somatic cells, chromatin integrity maintenance seems differentially regulated in the germline.

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