Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 µM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Ectopic pregnancy and epithelial to mesenchymal transition: is there a link?

Ectopic pregnancy (EP) is defined as the implantation of an embryo outside of the uterus and is a leading cause of first trimester maternal mortality and morbidity. This article discusses a possible role for epithelial to mesenchymal transition in the pathogenesis of EP, given the notable similarity of protein expression between the two processes.

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer.

BACKGROUND: Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues. METHODS: We developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24-240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified. RESULTS: Cisplatin exposure resulted in a significant reduction in the total number of germ cells (- 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (- 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (- 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure. CONCLUSIONS: This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

Comparative gonadotoxicity of the chemotherapy drugs cisplatin and carboplatin on prepubertal mouse gonads.

The treatment of childhood cancer with chemotherapy drugs can result in infertility in adulthood. Newer generations of drugs are developed to replace parent drugs, with the potential benefits of less toxic side effects. For platinum alkylating-like drugs, in contrast to the parent compound cisplatin, the newer-generation drug carboplatin is reported to have reduced toxicity in some respects, despite being administered at 5-15 times higher than the cisplatin dose. Whether carboplatin is also less toxic than cisplatin to the reproductive system is unknown. Here we compare the gonadotoxic impact of cisplatin and carboplatin on female and male mouse prepubertal gonads. In vitro cultured CD1 mouse ovaries or testis fragments were exposed to either cisplatin or carboplatin for 24 h on Day 2 of culture and analysed by Day 6. A dose response for each drug was determined for the ovary (0.5, 1 & 5 µg/ml cisplatin and 1, 5 & 10 µg/ml carboplatin) and the testis (0.01, 0.05 & 0.1 µg/ml cisplatin and 0.1, 0.5 & 1 µg/ml carboplatin). For the ovary, unhealthy follicles were evident from 1 µg/ml cisplatin (73% unhealthy, P = 0.001) and 5 µg/ml carboplatin (84% unhealthy, P = 0.001), with a concomitant reduction in follicle number (P = 0.001). For the testis, the proliferating germ cell population was significantly reduced from 0.05 µg/ml cisplatin (73% reduction, P = 0.001) and 0.5 µg/ml carboplatin (75% reduction, P = 0.001), with no significant impact on the Sertoli cell population. Overall, results from this in vitro animal model study indicate that, at patient equivalent concentrations, carboplatin is no less gonadotoxic than cisplatin.

Single and combined effects of cisplatin and doxorubicin on the human and mouse ovary in vitro.

Chemotherapy drugs are administered to patients using combination regimens, and as such the possibility of multiplicative effects between drugs need to be investigated. This study examines the individual and combined effects of the chemotherapy drugs cisplatin and doxorubicin on the human ovary. Although cisplatin and doxorubicin are known to affect female fertility, there is limited information about their direct effects on the human ovary, and none examining the possibility of combined, multiplicative effects of co-exposure to these drugs. Here, human ovarian biopsies were obtained from 14 women at the time of caesarean section, with 38 mouse ovaries also obtained from neonatal C57Bl/6J mice. Tissue was cultured for 6 days prior to analyses, with chemotherapy drugs added to culture medium on the second day of culture only. Treatment groups of a single (5 µg/mL human; 0.5 µg/mL mouse) or double (10 µg/mL human; 1.0 µg/mL mouse) dose of cisplatin, a single (1 µg/mL human; 0.05 µg/mL mouse) or double (2 µg/mL human; 0.01 µg/mL mouse) dose of doxorubicin or a combination of a single dose of both drugs together were compared to controls without drug exposure. Exposure to cisplatin or doxorubicin significantly decreased follicle health in human and mouse, supporting the suitability of the mouse as a model for the human ovary. There was also a significant reduction of mouse follicle number. Human ovarian stromal tissue exhibited increased apoptosis and decreased cell proliferation. Crucially, there was no evidence indicating the occurrence of multiplicative effects between cisplatin and doxorubicin.

How does chemotherapy treatment damage the prepubertal testis?

Chemotherapy treatment is a mainstay of anticancer regimens, significantly contributing to the recent increase in childhood cancer survival rates. Conventional cancer therapy targets not only malignant but also healthy cells resulting in side effects including infertility. For prepubertal boys, there are currently no fertility preservation strategies in use, although several potential methods are under investigation. Most of the current knowledge in relation to prepubertal gonadotoxicity has been deduced from adult studies; however, the prepubertal testis is relatively quiescent in comparison to the adult. This review provides an overview of research to date in humans and animals describing chemotherapy-induced prepubertal gonadotoxicity, focusing on direct gonadal damage. Testicular damage is dependent upon the agent, dosage, administration schedule and age/pubertal status at time of treatment. The chemotherapy agents investigated so far target the germ cell population activating apoptotic pathways and may also impair Sertoli cell function. Due to use of combined chemotherapy agents for patients, the impact of individual drugs is hard to define, however, use of in vivo and in vitro animal models can overcome this problem. Furthering our understanding of how chemotherapy agents target the prepubertal testis will provide clarity to patients on the gonadotoxicity of different drugs and aid in the development of cytoprotective agents.

Maturational gene upregulation and mitochondrial activity enhancement in mouse in vitro matured oocytes and using granulosa cell conditioned medium.

SummaryThe high miscarriage rates that result following transfer of embryos derived from in vitro maturation (IVM) of oocytes necessitate improvements in the processes involved. This study aimed to improve the quality of in vitro matured oocytes using granulosa cell conditioned medium (GCCM) as the culture medium. In this work, germinal vesicle (GV)-stage oocytes from NMRI mice were collected and cultured using three types of culture medium: Base medium (BM) (control), 50% granulosa cell conditioned medium (GCCM50) and 100% GCCM (GCCM100). After IVM, the mitochondria activity potential and viability of metaphase II (MII) oocytes were evaluated by JC-1 and trypan blue staining, respectively. Maturational gene expression levels of CyclinB1, Cdk1 and Gdf9 in the control, GCCM50 and GCCM100 samples were analyzed using real-time polymerase chain reaction (PCR). The viability rate of in vitro matured oocytes was highest in the GCCM50 group. JC-1 staining showed that GCCM50 enhances mitochondrial activity more than the other groups (P < 0.05). Gene expression levels of Cdk1 and Gdf9 were higher in the group with GCCM50 treatment, than in the control and GCCM100 groups (P < 0.05), while the expression level of CyclinB1 did not differ among the groups. The results indicated that a 50% concentration of GCCM in combination with BM components enhanced MII and viability rates and mitochondria activity of mouse immature oocytes.

Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice.

STUDY QUESTION: Does the Irinotecan metabolite 7-ethyl-10-hydroxycamptothecan (SN38) damage the gonads of male and female prepubertal mice? SUMMARY ANSWER: The Irinotecan metabolite SN38 reduces germ cell numbers within the seminiferous tubules of mouse testes at concentrations that are relevant to cancer patients, while in contrast it has little if any effect on the female germ cell population. WHAT IS KNOWN ALREADY: Little is known about the role of the chemotherapeutic agent Irinotecan on female fertility, with only one article to date reporting menopausal symptoms in perimenopausal women treated with Irinotecan, while no data are available either on adult male fertility or on the impact of Irinotecan on the subsequent fertility of prepubertal cancer patients, female or male. STUDY DESIGN SIZE, DURATION: Male and female gonads were obtained from postnatal day 5 C57BL/6 mice and exposed in vitro to a range of concentrations of the Irinotecan metabolite SN38: 0.002, 0.01, 0.05, 0.1 or 1 µg ml-1 for the testis and 0.1, 1, 2.5 or 5 µg ml-1 for the ovary, with treated gonads compared to control gonads not exposed to SN38. SN38 was dissolved in 0.5% dimethyl sulfoxide, with controls exposed to the same concentration of diluent. The number of testis fragments used for each analysis ranged between 3 and 9 per treatment group, while the number of ovaries used for each analysis ranged between 4 and 12 per treatment group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Neonatal mouse gonads were developed in vitro, with tissue analysed at the end of the 4-6 day culture period, following immunofluorescence or hematoxylin and eosin staining. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis test followed by Dunns post-test for non-parametric data. MAIN RESULTS AND THE ROLE OF CHANCE: Abnormal testis morphology was observed when tissues were exposed to SN38, with a smaller seminiferous tubule diameter at the highest concentration of SN38 (1 µg ml-1, p < 0.001 versus control) and increased number of Sertoli cell-only tubules at the two highest concentrations of SN38 (0.1 µg ml-1, p < 0.001; 1 µg ml-1, p < 0.0001, both versus control). Within seminiferous tubules, a dose response decrease was observed in both germ cell number (mouse vasa homologue (MVH)-positive cells) and in proliferating cell number (bromodeoxyuridine (BrdU)-positive cells), with significance reached at the two highest concentrations of SN38 (0.1 µg ml-1, p < 0.01 for both; 1 µg ml-1, p < 0.001-MVH, p < 0.01-BrdU; all versus control). No change was seen in protein expression of the apoptotic marker cleaved caspase 3. Double immunofluorescence showed that occasional proliferating germ cells were present in treated testes, even after exposure to the highest drug concentration. When prepubertal ovaries were treated with SN38, no effect was seen on germ cell number, apoptosis or cell proliferation, even after exposure to the highest drug concentrations. LIMITATIONS REASONS FOR CAUTION: As with any study using in vitro experiments with an experimental animal model, caution is required when extrapolating the present findings to humans. Differences between human and mouse spermatogonial development also need to be considered when assessing the effect of chemotherapeutic exposure. However, the prepubertal testes and ovaries used in the present studies contain germ cell populations that are representative of those found in prepubertal patients, and experimental tissues were exposed to drug concentrations within the range found in patient plasma. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the prepubertal mouse ovary is relatively insensitive to exposure to the Irinotecan metabolite SN38, while it induces a marked dose-dependent sensitivity in the testicular germ cell population. The study identifies the importance of further investigation to identify the risk of infertility in young male cancer patients treated with Irinotecan. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: Work supported by Medical Research Grant (MRC) grant G1002118 and Children with Cancer UK grant 15-198. The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research.

Etoposide damages female germ cells in the developing ovary.

BACKGROUND: As with many anti-cancer drugs, the topoisomerase II inhibitor etoposide is considered safe for administration to women in the second and third trimesters of pregnancy, but assessment of effects on the developing fetus have been limited. The purpose of this research was to examine the effect of etoposide on germ cells in the developing ovary. Mouse ovary tissue culture was used as the experimental model, thus allowing us to examine effects of etoposide on all stages of germ cell development in the same way, in vitro. RESULTS: Fetal ovaries from embryonic day 13.5 CD1 mice or neonatal ovaries from postnatal day 0 CD1 mice were cultured with 50-150 ng ml(-1) or 50-200 ng ml(-1) etoposide respectively, concentrations that are low relative to that in patient serum. When fetal ovaries were treated prior to follicle formation, etoposide resulted in dose-dependent damage, with 150 ng ml(-1) inducing a near-complete absence of healthy follicles. In contrast, treatment of neonatal ovaries, after follicle formation, had no effect on follicle numbers and only a minor effect on follicle health, even at 200 ng ml(-1). The sensitivity of female germ cells to etoposide coincided with topoisomerase IIα expression: in the developing ovary of both mouse and human, topoisomerase IIα was expressed in germ cells only prior to follicle formation. CONCLUSIONS: Exposure of pre-follicular ovaries, in which topoisomerase IIα expression was germ cell-specific, resulted in a near-complete elimination of germ cells prior to follicle formation, with the remaining germ cells going on to form unhealthy follicles by the end of culture. In contrast, exposure to follicle-enclosed oocytes, which no longer expressed topoisomerase IIα in the germ cells, had no effect on total follicle numbers or health, the only effect seen specific to transitional follicles. Results indicate the potential for adverse effects on fetal ovarian development if etoposide is administered to pregnant women when germ cells are not yet enclosed within ovarian follicles, a process that starts at approximately 17 weeks gestation and is only complete towards the end of pregnancy.

Docetaxel induces moderate ovarian toxicity in mice, primarily affecting granulosa cells of early growing follicles.

Advances in cancer therapy have focused attention on the quality of life of cancer survivors. Since infertility is a major concern following chemotherapy, it is important to characterize the drug-specific damage to the reproductive system to help find appropriate protective strategies. This study investigates the damage on neonatal mouse ovary maintained in vitro for 6 days, and exposed for 24 h (on Day 2) to clinically relevant doses of Docetaxel (DOC; low: 0.1 µM, mid: 1 µM, high: 10 µM). Furthermore, the study explores the putative protective action exerted by Tri-iodothyronine (T3; 10(-7) M). At the end of culture, morphological analyses and follicle counts showed that DOC negatively impacts on early growing follicles, decreasing primary follicle number and severely affecting health at the transitional and primary stages. Poor follicle health was mainly due to effects on granulosa cells, indicating that the effects of DOC on oocytes were likely to be secondary to granulosa cell damage. DOC damages growing follicles specifically, with no direct effect on the primordial follicle reserve. Immunostaining and western blotting showed that DOC induces activation of intrinsic, type II apoptosis in ovarian somatic cells; increasing the levels of cleaved caspase 3, cleaved caspase 8, Bax and cleaved poly(ADP-ribose) polymerase, while also inducing movement of cytochrome C from mitochondria into the cytosol. T3 did not prevent the damage induced by the low dose of DOC. These results demonstrated that DOC induces a gonadotoxic effect on the mouse ovary through induction of somatic cell apoptosis, with no evidence of direct effects on the oocyte, and that the damaging effect is not mitigated by T3.

Spermatogonial quantity in prepubertal boys undergoing fertility preservation is comparable between haematological and non-haematological cancers.

Fertility restoration potential of immature testicular tissue (ITT) depends on the number of spermatogonial cells in the retrieved tissue prior to cryopreservation in oncofertility programme. There are limited data on the association between type of malignancy and testicular germ cell population. Hence, this study is aimed to investigate the spermatogonial and Sertoli cell population in ITT retrieved from 14 pre-pubertal boys who opted for fertility preservation. Histopathological and immunochemical analysis of seminiferous tubules from haematological (N = 7) and non-haematological (N = 7) malignant patients revealed 3.43 ± 2.92 and 1.71 ± 1.81 spermatogonia per tubular cross section (S/T), respectively. The Sertoli cell number was comparable between haematological and non-haematological group (18.42 ± 3.78 and 22.03 ± 10.43). Spermatogonial quantity in ITT did not vary significantly between haematological and non-haematological cancers. This observation, though preliminary, would contribute to the limited literature on paediatric male oncofertility.

Implantation failure in mice with a disruption in Phospholipase C beta 1 gene: lack of embryonic attachment, aberrant steroid hormone signalling and defective endocannabinoid metabolism.

Phospholipase C beta 1 (PLCß1) is a downstream effector of G-protein-coupled receptor signalling and holds central roles in reproductive physiology. Mice with a disruption in the Plcß1 gene are infertile with pleiotropic reproductive defects, the major reproductive block in females being implantation failure. Here, PLCß1 was demonstrated at the luminal and glandular epithelia throughout the pre- and peri-implantation period, with transient stromal expression during 0.5-1.5 days post coitum (dpc). Examination of implantation sites at 4.5 dpc showed that in females lacking functional PLCß1 (knock-out (KO) females), embryos failed to establish proper contact with the uterine epithelium. Proliferating luminal epithelial cells were evident in KO implantation sites, indicating failure to establish a receptive uterus. Real-time PCR demonstrated that KO implantation sites had aberrant ovarian steroid signalling, with high levels of estrogen receptor α, lactoferrin and amphiregulin mRNA, while immunohistochemistry revealed very low levels of estrogen receptor α protein, possibly due to rapid receptor turnover. KO implantation sites expressed markedly less fatty acid amide hydrolase and monoacylglycerol lipase, indicating that endocannabinoid metabolism was also affected. Collectively, our results show that PLCß1 is essential for uterine preparation for implantation, and that defective PLCß1-mediated signalling during implantation is associated with aberrant ovarian steroid signalling and endocannabinoid metabolism.

Identification of cells migrating from the thecal layer of ovarian follicles.

In the mammalian ovary, oocytes are contained within ovarian follicles. These consist in an oocyte surrounded by supporting cells: an inner layer of granulosa cells and an outer layer of thecal cells separated by a basal lamina. At any one time, a developing cohort of follicles exists, from which only a small species-specific number are selected for continued development towards ovulation, with the remainder dying by follicular atresia. Here, we use in vitro methods to study interactions between two follicles in culture (follicle co-cultures). We show that, when two individual follicles are grown together in culture, cells and cellular processes migrate from the outer thecal layer of one follicle to the thecal layer of the other co-cultured follicle. These cells are identified as a mixed population containing primarily endothelial but also neuronal cells. Both are able to migrate through the ovarian interstitum, making contact with the basal lamina of other follicles and with similar cells from these other follicles. Networks of such cells might be involved in interfollicular communication and in the coordination of follicle selection for ovulation.

The impact of vincristine on testicular development and function in childhood cancer.

BACKGROUND: Increasing childhood cancer survival rates in recent decades have led to an increased focus on fertility as a long-term complication of cancer treatment. Male childhood cancer survivors often face compromised testicular function as a late effect of chemotherapy exposure, with no well-established options to prevent such damage and subsequent infertility. Despite vincristine being considered to be associated with low-gonadotoxic potential, in prepubertal rodents, it was recently shown to result in morphological alterations of the testis and in severely impaired fertility. OBJECTIVE AND RATIONALE: This systematic review aimed to evaluate the effects of vincristine-containing regimens on human prepubertal testis with reference to testicular function and fertility in adulthood. SEARCH METHODS: The systematic search of the literature was conducted according to PRISMA guidelines, and the study was registered with PROSPERO. PubMed and Scopus were searched for articles published in English between 01 January 1900 and 05 March 2021, with the search including 'chemotherapy', 'vincristine', 'prepubertal', 'testis', 'spermatogenesis' and related terms. Abstracts and full-text articles were screened and selected for, providing they met the inclusion criteria (≤12 years at treatment, exposure to vincristine-containing regimens and long-term fertility outcomes). Additional studies were identified via bibliography screening. Bias evaluation across included studies was conducted using the ROBINS-I tool, subdivided into assessment for confounding, participant selection, intervention classification, missing data, outcome measurements and selection of reported results. OUTCOMES: Our initial search identified 288 articles of which 24 (8%; n = 7134 males) met all inclusion criteria. Control groups were included for 9/24 (38%) studies and 4/24 (17%) studies provided sub-analysis of the relative gonadotoxicity of vincristine-based agents. Primary outcome measures were: fertility and parenthood; semen analysis (World Health Organization criteria); and hormonal function and testicular volume. For the studies that performed vincristine sub-analysis, none reported negative associations with vincristine for the potential of siring a pregnancy, including the largest (n = 6224; hazard ratio = 0.56) controlled study. For semen analysis, no significant difference versus healthy controls was illustrated for mitotic inhibitors (including vincristine) following sub-analysis in one study (n = 143). For hormone analysis, a single study did not find significant impacts on spermatogenesis attributed to vincristine based on levels of FSH and semen analysis, which meant that its administration was unlikely to be responsible for the diminished testicular reserve; however, most of the studies were based on low numbers of patients receiving vincristine-containing chemotherapy. Analysis of bias demonstrated that studies which included vincristine exposure sub-analysis had a lower risk of bias when compared with cohorts which did not. WIDER IMPLICATIONS: In contrast to recent findings in rodent studies, the limited number of clinical studies do not indicate gonadotoxic effects of vincristine following prepubertal exposure. However, given the relative lack of data from studies with vincristine sub-analysis, experimental studies involving vincristine exposure using human testicular tissues are warranted. Results from such studies could better inform paediatric cancer patients about their future fertility and eligibility for fertility preservation before initiation of treatment.

Recent advances in the understanding of tubal ectopic pregnancy.

Ectopic pregnancy (EP) is described as the implantation of an embryo outside the normal uterine cavity. It most commonly occurs in the fallopian tube, hence termed a tubal ectopic pregnancy (tEP). It is a gynaecological emergency and remains the leading cause of direct maternal mortality related to the first trimester of pregnancy worldwide. This article explores the emergence of additional risk factors for tEP, showing new evidence for identifying patient risk factors and highlighting potential areas of research. Additionally, we discuss the up-to-date patient-centred approach for the diagnosis, management and counselling of patients with tEP and ongoing clinical trials for the improvement of medical management.

Oncofertility awareness among primary care physicians in India.

BACKGROUND: Primary care physicians not only coordinate referrals to oncology services but can play a crucial role in successful fertility preservation referrals in cancer-diagnosed patients. Hence, it is important to assess their knowledge and attitudes towards fertility preservation. METHODS: An eighteen-item oncofertility survey was administered to primary care physicians between May 2019 to September 2020.  Results: A total of forty-six responses were received and analysed. About 60% of primary care physicians did not have adequate knowledge about available fertility preservation options and only 26-32% were aware of international guidelines recommending fertility preservation in cancer patients.  Conclusions: Imparting awareness and knowledge of fertility preservation and its options to primary care physicians could enable an integrated cancer care model while also facilitating successful oncofertility referrals in countries like India.

Cisplatin Effects on the Human Fetal Testis - Establishing the Sensitive Period for (Pre)Spermatogonial Loss and Relevance for Fertility Preservation in Pre-Pubertal Boys.

Background: Exposure to chemotherapy during childhood can impair future fertility. Studies using in vitro culture have shown exposure to platinum-based alkylating-like chemotherapy reduces the germ cell number in the human fetal testicular tissues. We aimed to determine whether effects of exposure to cisplatin on the germ cell sub-populations are dependent on the gestational age of the fetus and what impact this might have on the utility of using human fetal testis cultures to model chemotherapy exposure in childhood testis. Methods: We utilised an in vitro culture system to culture pieces of human fetal testicular tissues (total n=23 fetuses) from three different gestational age groups (14-16 (early), 17-19 (mid) and 20-22 (late) gestational weeks; GW) of the second trimester. Tissues were exposed to cisplatin or vehicle control for 24 hours, analysing the tissues 72 and 240 hours post-exposure. Number of germ cells and their sub-populations, including gonocytes and (pre)spermatogonia, were quantified. Results: Total germ cell number and number of both germ cell sub-populations were unchanged at 72 hours post-exposure to cisplatin in the testicular tissues from fetuses of the early (14-16 GW) and late (20-22 GW) second trimester. In the testicular tissues from fetuses of mid (17-19 GW) second trimester, total germ cell and gonocyte number were significantly reduced, whilst (pre)spermatogonial number was unchanged. At 240 hours post-exposure, the total number of germ cells and that of both sub-populations was significantly reduced in the testicular tissues from fetuses of mid- and late-second trimester, whilst germ cells in early-second trimester tissues were unchanged at this time-point. Conclusions: In vitro culture of human fetal testicular tissues can be a useful model system to investigate the effects of chemotherapy-exposure on germ cell sub-populations during pre-puberty. Interpretation of the results of such studies in terms of relevance to later (infant and pre-pubertal) developmental stages should take into account the changes in germ cell composition and periods of germ cell sensitivity in the human fetal testis.

Maintenance of Sertoli Cell Number and Function in Immature Human Testicular Tissues Exposed to Platinum-Based Chemotherapy-Implications for Fertility Restoration.

Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts of exposure to alkylating chemotherapy on future fertility. We recently demonstrated germ cell loss in immature human testicular tissues following exposure to platinum-based chemotherapeutic drugs. This study investigated the effects of platinum-based chemotherapy exposure on the somatic Sertoli cell population in human fetal and pre-pubertal testicular tissues. Methods: Human fetal (n = 23; 14-22 gestational weeks) testicular tissue pieces were exposed to cisplatin (0.5 or 1.0 µg/ml) or vehicle for 24 h in vitro and analysed 24-240 h post-exposure or 12 weeks after xenografting. Human pre-pubertal (n = 10; 1-12 years) testicular tissue pieces were exposed to cisplatin (0.5 µg/ml), carboplatin (5 µg/ml) or vehicle for 24 h in vitro and analysed 24-240 h post-exposure; exposure to carboplatin at 10-times the concentration of cisplatin reflects the relative clinical doses given to patients. Immunohistochemistry was performed for SOX9 and anti-Müllerian hormone (AMH) expression and quantification was carried out to assess effects on Sertoli cell number and function respectively. AMH and inhibin B was measured in culture medium collected post-exposure to assess effects on Sertoli cell function. Results: Sertoli cell (SOX9+ve) number was maintained in cisplatin-exposed human fetal testicular tissues (7,647 ± 459 vs. 7,767 ± 498 cells/mm2; p > 0.05) at 240 h post-exposure. No effect on inhibin B (indicator of Sertoli cell function) production was observed at 96 h after cisplatin (0.5 and 1.0 µg/ml) exposure compared to control (21 ± 5 (0.5 µg/ml cisplatin) vs. 23 ± 7 (1.0 µg/ml cisplatin) vs. 25 ± 7 (control) ng/ml, p > 0.05). Xenografting of cisplatin-exposed (0.5 µg/ml) human fetal testicular tissues had no long-term effect on Sertoli cell number or function (percentage seminiferous area stained for SOX9 and AMH, respectively), compared with non-exposed tissues. Sertoli cell number was maintained in human pre-pubertal testicular tissues following exposure to either 0.5 µg/ml cisplatin (6,723 ± 1,647 cells/mm2) or 5 µg/ml carboplatin (7,502 ± 627 cells/mm2) compared to control (6,592 ± 1,545 cells/mm2). Conclusions: This study demonstrates maintenance of Sertoli cell number and function in immature human testicular tissues exposed to platinum-based chemotherapeutic agents. The maintenance of a functional Sertoli cell environment following chemotherapy exposure suggests that fertility restoration by spermatogonial stem cell (SSC) transplant may be possible in boys facing platinum-based cancer treatment.

Decreased oocyte DAZL expression in mice results in increased litter size by modulating follicle-stimulating hormone-induced follicular growth.

While the germ cell-specific RNA binding protein, DAZL, is essential for oocytes to survive meiotic arrest, DAZL heterozygous (het) mice have an increased ovulation rate that is associated with elevated inhibin B and decreased plasma follicle-stimulating hormone (FSH). The relationship between decreased oocyte DAZL expression and enhanced follicular development in het mice was investigated using in vitro follicle cultures and in vivo modulation of endogenous FSH, by treating mice with inhibin and exogenous FSH. In vitro, follicles from het mice are more sensitive to FSH than those of wild-type (wt) mice and can grow in FSH concentrations that are deleterious to wild-type follicles. In vivo, despite no differences between genotypes in follicle population profiles, analysis of granulosa cell areas in antral follicles identified a significantly greater number of antral follicles with increased granulosa cell area in het ovaries. Modulation of FSH in vivo, using decreasing doses of FSH or ovine follicular fluid as a source of inhibin, confirmed the increased responsiveness of het antral follicles to FSH. Significantly more follicles expressing aromatase protein confirmed the earlier maturation of granulosa cells in het mice. In conclusion, it is suggested that DAZL expression represses specific unknown genes that regulate the response of granulosa cells to FSH. If this repression is reduced, as in DAZL het mice, then follicles can grow to the late follicular stage despite declining levels of circulating FSH, thus leading to more follicles ovulating and increased litter size.

Differential expression and regulation by activin of the neurotrophins BDNF and NT4 during human and mouse ovarian development.

The tropomyosin-related kinase (Trk) B neurotrophin receptor is essential for ovarian germ cell survival and primordial follicle formation, but the contributions of its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), are unknown. We have investigated their expression and regulation in developing human and mouse ovaries. BDNF expression increased with increasing gestation, expression of human NTF4 and of both Ntf5 and Bdnf in the mouse was unchanged. Bdnf expression was dramatically lower than Ntf5 in the mouse, but levels were comparable in the human. Human fetal ovarian somatic cells expressed BDNF. Activin A selectively regulated BDNF and Ntf5 expression in human and mouse, respectively, identifying an oocyte/somatic signaling pathway which might mediate the pro-survival effects of activin. These data reveal that expression and regulation of the TrkB ligands are differentially controlled in the developing ovaries of humans and mice, and identify BDNF as a potential regulator of germ cell fate in the human fetal ovary.