Micro RNA in Semen/Urine from Non-Obstructive Azoospermia Patients as Biomarkers to Predict the Presence of Testicular Spermatozoa and Spermatogonia.

About half of testicular sperm extraction (TESE) procedures in men with non-obstructive azoospermia (NOA), including men with Klinefelter syndrome (KS), are unsuccessful. To avoid unnecessary invasive surgery, biomarkers for spermatozoa were studied. In addition, markers for spermatogonia in testis tissue were explored. This study aimed to find biomarkers in the semen and/or urine of NOA patients to predict the presence of spermatogonia in the testis. Differentially expressed miRNAs were identified (1) between samples from patients with and without a positive TESE procedure as well as (2) between TESE-negative patients with and without spermatogonia. A total of thirteen upregulated miRNAs (ten in seminal plasma and three in urine) were found in the TESE-negative/spermatogonia-positive group compared to the TESE-negative/spermatogonia-negative group. These miRNAs could be potential biomarkers for spermatogonia; however, more research is necessary to validate their predictive power.

Transcriptomic differences between fibrotic and non-fibrotic testicular tissue reveal possible key players in Klinefelter syndrome-related testicular fibrosis.

Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most men with KS suffer from an early germ cell loss and testicular fibrosis from puberty onwards. Mechanisms responsible for these processes remain unknown. Previous genomics studies on testis tissue from men with KS focused on germ cell loss, while a transcriptomic analysis focused on testicular fibrosis has not yet been performed. This study aimed to identify factors involved in the fibrotic remodelling of KS testes by analysing the transcriptome of fibrotic and non-fibrotic testicular tissue. RNA sequencing was performed to compare the genes expressed in testicular samples with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) fibrosis (n = 5, each). Additionally, differentially expressed genes (DEGs) between KS and testis atrophy samples were studied to reveal KS-specific fibrotic genes. DEGs were considered significant when p < 0.01 and log2FC > 2. Next, downstream analyses (GO and KEGG) were performed. Lastly, RNA in situ hybridization was performed to validate the results. The first analysis (fibrotic vs non-fibrotic) resulted in 734 significant DEGs (167 up- and 567 down-regulated). Genes involved in the extracellular structure organization (e.g. VCAM1) were found up-regulated. KEGG analysis showed an up-regulation of genes involved in the TGF-ß pathway. The KS vs testis atrophy analysis resulted in 539 significant DEGs (59 up- and 480 down-regulated). Chronic inflammatory response genes were found up-regulated. The overlap of X-linked DEGs from the two analyses revealed three genes: matrix-remodelling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). RNA in situ hybridization showed an overexpression of VCAM1, MXRA5 and DCX within the fibrotic group compared with the non-fibrotic group. To summarize, this study revealed DEGs between fibrotic and non-fibrotic testis tissue, including VCAM1. In addition, X-linked fibrotic genes were revealed, e.g. MXRA5, DCX and VCX3B. Their potential role in KS-related testicular fibrosis needs further study.

Testicular biopsy for fertility preservation in early-diagnosed Klinefelter patients: patient characteristics and long-term follow-up.

RESEARCH QUESTION: Which early-diagnosed Klinefelter syndrome patients have been offered cryopreservation of testicular tissue as part of fertility preservation before spermatogonial stem cell (SSC) loss? Do these Klinefelter syndrome patients present with behavioural, cognitive and/or psychological problems? Does a testicular biopsy procedure have long-term effects on the gonadal development of Klinefelter syndrome patients? DESIGN: Early-diagnosed Klinefelter syndrome patients followed between 2009 and 2020 and offered testicular tissue banking in an experimental context at the Universitair Ziekenhuis Brussel were included. The prevalence of behavioural, cognitive and/or psychological problems was determined. Changes in testicular volume and in gonadal function (LH, FSH, testosterone and inhibin B [INHB]) were studied. RESULTS: Of the 48 Klinefelter syndrome patients included, 22 had testicular tissue removed (biopsy group) and 26 had no surgical intervention (control group). The need for specialized education was significantly higher in prenatally (P = 0.0159) and prepubertally (P = 0.0002) diagnosed Klinefelter syndrome patients. Psychological problems were significantly more prevalent in Klinefelter syndrome patients who did not opt for fertility preservation (P = 0.0447). In the first 4.2 (1.9-9.1) years after testicular biopsy, no difference in testicular volume was observed between the biopsied and the contralateral non-biopsied testes (P > 0.9999). After pubertal onset, no differences in LH, FSH, testosterone and INHB were found between the biopsy and the control groups (P = 0.1324 for LH, P > 0.9999 for FSH, P = 0.5433 for testosterone, P > 0.9999 for INHB). CONCLUSION: Early-diagnosed Klinefelter syndrome patients presented with behavioural, cognitive and/or psychological problems. Only psychological problems seemed to influence the decision towards fertility preservation. Follow-up data confirm that harvesting testicular tissue does not have a long-term impact on the gonadal development of Klinefelter syndrome patients.

Intratesticular xenografting of Klinefelter pre-pubertal testis tissue as potential model to study testicular fibrosis.

RESEARCH QUESTION: Is intratesticular xenotransplantation a potential ex-vivo model for studying testicular fibrosis related to Klinefelter syndrome? STUDY DESIGN: First, a feasibility study of an ex-vivo model to study testicular fibrosis in patients with Klinefelter syndrome was performed. Testis tissue from boys with pre-pubertal Klinefelter syndrome (n = 3) and controls (n = 2) (<18 years) was grafted to the mouse testis (n = 12) and recovered after 2, 4, 6 and 8 weeks. Part two of this study consisted of a validation of this model, evaluating the effects of the mast cell blocker ketotifen on the histology of the grafts of Klinefelter syndrome (n = 5) and controls (n = 3), transplanted to mice (n = 10), after 4 weeks of ketotifen or saline treatment. Immunohistochemistry determined the histology of the grafts and the presence of mast cells and spermatogonia. RESULTS: The feasibility study showed that 4 weeks after transplantation, all Klinefelter syndrome grafts could be recovered. Later, degeneration was observed. Most recovered grafts showed an intact histology, with 67 ± 12% intact tubules for the Klinefelter syndrome grafts and 65 ± 15% of intact tubules for the control grafts. In the few remaining Klinefelter syndrome grafts, treatment with ketotifen improved testicular histology compared with non-treated grafts. Graft survival was patient dependent. No germ cell loss was observed after transplantation. CONCLUSION: Xenografting could become a model for the longitudinal study of the fibrotic process related to Klinefelter syndrome; however, the current model has a limited survival period and patient-specific differences in histology.

Low Testosterone and Semen Parameters in Male Partners of Infertile Couples Undergoing IVF with a Total Sperm Count Greater than 5 Million.

Low serum testosterone is found in approximately 15% of subfertile men. Although testosterone is essential in spermatogenesis, it is unclear whether low testosterone levels may have a negative impact on the semen parameters of men belonging to infertile couples with a total sperm count greater than 5 million. Furthermore, it is debatable whether the initial evaluation of the subfertile male should include an endocrine assessment. This was a retrospective, single-center cohort study conducted at a tertiary fertility clinic. Male partners of infertile couples undergoing in vitro fertilization (IVF), with a total sperm count greater than 5 million, were included. All men provided morning blood samples, and none had been on exogenous testosterone or other relevant medications. Low total testosterone (TT) was defined as <264 ng/dL. Free T was calculated using TT and sex hormone-binding globulin (SHBG) levels (nmol/L) by a constant albumin concentration of 43 g/L. In total, 853 patients were included: 116 had low TT (<264 ng/dL) and 737 had normal TT (≥264 ng/dL). Semen volume, sperm cell count, progressive (A + B) motility and morphology (≥4% strict Kruger) were lower in the low TT group but not significantly different between low and normal TT groups (3.2 ± 1.79 vs. 3.23 ± 1.64, p = 0.87; 76.82 ± 83.18 vs. 67.55 ± 57.70, p = 0.7; 54.89 ± 19.45 vs. 56.25 ± 19.03, p = 0.6; 5.77 ± 3.23 vs. 6.89 ± 3.94, p = 0.23). The percentage of patients with below-reference sperm volume (<1.5 mL), cell count (<15 × 106/mL), motility (A + B) (<32%) and morphology (<4%) was higher in the low TT group but not statistically different compared to the normal TT group. Multivariable regression analysis revealed that low TT and free T levels had no significant effect on the aforementioned semen parameters (coefficient: 3.94, 0.88, 1.37, 0.39; p = 0.53, 0.8, 0.3, 0.2; coefficient: 0.001, 0.06, 0.007, 0.0002; p = 0.73, 0.52, 0.85, 0.98). Despite our robust methodological approach, the presence of biases related to retrospective design cannot be excluded. Our findings highlighted the lack of association between low TT levels and semen parameter alterations in male partners of infertile couples undergoing IVF, with a total sperm count greater than 5 million. However, it is important to emphasize that more patients in the low TT group had subnormal semen parameters, albeit the difference was not statistically significant. Larger, prospective studies are warranted in order to validate these findings, as well as to investigate the existence of a TT threshold below which semen parameters might be negatively affected.

Testicular immune cells and vasculature in Klinefelter syndrome from childhood up to adulthood.

STUDY QUESTION: Is the distribution of immune cells and the testicular vasculature altered in testicular biopsies from patients with Klinefelter syndrome (KS)? SUMMARY ANSWER: Increased numbers of macrophages and mast cells, an increased expression of decorin and an increased blood vessel density were found in KS samples compared to controls. WHAT IS KNOWN ALREADY: Most KS patients are infertile due to an early germ cell loss. From puberty onwards, testicular fibrosis can be detected. How this fibrotic process is initiated remains unknown. STUDY DESIGN, SIZE, DURATION: In this study, the number of macrophages, mast cells and their secretory products were evaluated in KS, Sertoli cell only (SCO) and control patient samples. The association between immune cell numbers and level of fibrosis in KS tissue was examined. In addition, the vascularization within these testicular tissue biopsies was studied. For immunohistochemical evaluation, KS patients at different stages of testicular development were included: prepubertal (aged 4-7 years; n = 4), peripubertal (aged 11-17 years; n = 21) and adult (aged >18 years; n = 37) patients. In addition, testicular tissue biopsies of adult SCO (n = 33) and control samples for the three KS age groups (prepubertal n = 9; peripubertal n = 5; adult n = 25) were analysed. Gene expression analysis was performed on adult testicular tissue from KS (n = 5), SCO (n = 5) and control (n = 5) patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult (>18 years) KS, SCO and control testicular tissue biopsies were obtained during a testicular sperm extraction procedure. KS peripubertal (11-18 years), prepubertal (<11 years) and age-matched control biopsies were obtained from the biobank of the university hospital. Immunohistochemistry was used to determine the tubular structure (H/PAS), the number of spermatogonia (MAGE-A4), macrophages (CD68) and mast cells (tryptase) and the blood vessel density (Von Willebrand factor). In addition, quantitative real-time polymerase chain reaction was used to determine the expression of secretory products of macrophages and mast cells (tryptase, tumour necrosis factor alpha and decorin). MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in the number of macrophages (P < 0.0001) and mast cells (P = 0.0008) was found in the peritubular compartment of testes of adult KS patients compared to control samples. However, no association between the number of immune cells and the degree of fibrosis was observed. In adult SCO samples, a significant increase was seen for peritubular macrophage (P < 0.0001) and mast cell (P < 0.0001) numbers compared to control samples. In the interstitial compartment, a significant increase in mast cell number was found in adult SCO samples compared to KS (P < 0.0001) and control (P < 0.0001) tissue. A significant difference (P = 0.0431) in decorin expression could be detected in adult KS compared to control patients. Decorin expression was mostly seen in the walls of the seminiferous tubules. When comparing the vascularization between KS patients and age-matched controls, a significant increase (P = 0.0081) in blood vessel density could be observed only in prepubertal KS testicular tissue. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: As controls for this study, testicular tissue biopsies of men who underwent a vasectomy reversal or orchiectomy were used, but these men may not represent fertile controls. In addition, a high variability in immune cell numbers, secretory products expression and number of blood vessels could be observed amongst all patient samples. WIDER IMPLICATIONS OF THE FINDINGS: Increased numbers of macrophages and mast cells have previously been described in non-KS infertile men. Our results show that these increased numbers can also be detected in KS testicular tissue. However, no association between the number of macrophages or mast cells and the degree of fibrosis in KS samples could be detected. Decorin has previously been described in relation to fibrosis, but it has not yet been associated with testicular fibrosis in KS. Our results suggest a role for this proteoglycan in the fibrotic process since an increased expression was observed in adult KS tissue compared to controls. Impaired vascularization in KS men was suggested to be responsible for the KS-related disturbed hormone levels. Our results show a significant difference in blood vessel density, especially for the smallest blood vessels, between prepubertal KS samples and age-matched controls. This is the first study to report differences between KS and control testicular tissue at prepubertal age. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by grants from the Vrije Universiteit Brussel (E.G.) and the scientific Fund Willy Gepts from the UZ Brussel (D.V.S.). D.V.S. is a post-doctoral fellow of the Fonds voor Wetenschappelijk Onderzoek (FWO; 12M2819N). No conflict of interest is declared for this research project.

Characterization of the stem cell niche components within the seminiferous tubules in testicular biopsies of Klinefelter patients.

OBJECTIVE: To characterize the tubular environment in testicular biopsy tissues from patients with Klinefelter syndrome (KS). DESIGN: Observational immunohistochemical study. SETTING: Academic research unit. PATIENT(S): Males with KS and controls at different developmental time points: fetal, prepubertal, peripubertal, and adult. INTERVENTION(S): Immunohistochemical analysis of testicular biopsies samples to characterize maturation of Sertoli cells and tubular wall components-peritubular myoid cells (PTMC) and extracellular matrix (ECM) proteins. MAIN OUTCOME MEASURE(S): Intensity of antimüllerian hormone staining; proportion of Sertoli cells expressing androgen receptor (AR); and expression of tubular wall markers as characterized by identifying abnormal staining patterns. RESULT(S): Decreased expression for alpha smooth muscle actin 2 (ACTA2) was observed in peripubertal and adult KS as well as in Sertoli cell only (SCO) patients. Altered expression patterns for all ECM proteins were observed in SCO and KS biopsy tissues compared with controls. Only for collagen I and IV were altered expression patterns observed between KS and SCO patients. In peripubertal samples, no statistically significant differences were observed in the maturation markers, but altered ECM patterns were already present in some samples. CONCLUSION(S): The role of loss of ACTA2 expression in PTMC in the disintegration of tubules in KS patients should be further investigated. Future research is necessary to identify the causes of testicular fibrosis in KS patients. If the mechanism behind this fibrotic process could be identified, this process might be altered toward increasing the chances of fertility in KS patients.

Creating a Global Community of Practice for Oncofertility.

Fertility preservation in the cancer setting, known as oncofertility, is a field that requires cross-disciplinary interaction between physicians, basic scientists, clinical researchers, ethicists, lawyers, educators, and religious leaders. Funded by the National Institutes of Health, the Oncofertility Consortium (OC) was formed to be a scientifically grounded, transparent, and altruistic resource, both intellectual and monetary, for building this new field of practice capable of addressing the unique needs of young patients with cancer. The OC has expanded its attention to include other nonmalignant conditions that can threaten fertility, and the work of the OC now extends around the globe, involving partners who together have created a community of shared effort, resources, and practices. The OC creates materials that are translated, disseminated, and amended by all participants in the field, and local programs of excellence have developed worldwide to accelerate the pace and improve the quality of oncofertility research and practice. Here we review the global oncofertility programs and the capacity building activities that strengthen these research and clinical programs, ultimately improving patient care.

Is genetic fatherhood within reach for all azoospermic Klinefelter men?

BACKGROUND: Multidisciplinary management of Klinefelter cases is now considered good clinical practice in order to ensure optimal quality of life. Reproductive performance of Klinefelter men is an important issue however literature in this domain is limited and prone to bias. STUDY DESIGN: This was a retrospective longitudinal cohort study performed at a tertiary referral University Centre for Reproductive Medicine and Genetics. One hundred thirty-eight non-mosaic azoospermic Klinefelter patients undergoing their first testicular biopsy (TESE) between 1994 and 2013, followed by intracytoplasmic sperm injection (ICSI) with fresh or frozen-thawed testicular sperm in the female partner, were followed-up longitudinally. The main outcome measure was cumulative live birth rate per Klinefelter patient embarking on TESE-ICSI. FINDINGS: In forty-eight men (48/138) sperm were successfully retrieved at the first TESE (34.8%). The mean age of the patients was 32.4 years. Younger age at first TESE was associated with a higher sperm retrieval rate (p<0.001). Overall 39 couples underwent 62 ICSI cycles and 13 frozen embryo transfer cycles resulting in in 20 pregnancies and 14 live birth deliveries (16 children). The mean age of the female partner was 28.1 years. The crude cumulative delivery rate after four ICSI cycles was 35.9%. Per intention-to-treat however, only 10.1% (14/138) of the Klinefelter men starting treatment succeeded in having their biologically own child(ren). CONCLUSION: Non-mosaic Klinefelter patients with azoospermia seeking treatment by TESE-ICSI should be counseled that by intention-to-treat the chance of retrieving sperm is fair, however only a minority will eventually father genetically own children.

Gonadotropin Releasing Hormone Agonists or Antagonists for Preimplantation Genetic Diagnosis (PGD)? A Prospective Randomised Trial.

BACKGROUND: The use of GnRH analogue medication is essential in reproductive medicine to avoid premature ovulation by pituitary suppression for the duration of ovarian stimulation by gonadotrophins. The type of pituitary suppression by either GnRH agonist analogues versus GnRH antagonist analogues may result in different embryological hence clinical results. Preimplantation genetic diagnosis is a subtype of IVF in which embryos are created for genetic diagnosis of hereditary disorders in order to avoid genetically affected children. Embryological quality hence ovarian stimulation in preimplantation genetic diagnosis is crucial as genetic selection will reduce the number of available embryos to a fraction of the total. OBJECTIVE: The aim of this study was to assess the efficiency of GnRH antagonist versus GnRH agonist treatment for pituitary suppression in ovarian stimulation for PGD, by proxy of number and quality of embryos at cleavage stage available for biopsy. METHOD: We conducted a prospective randomised controlled trial comparing pituitary suppression by GnRH antagonist versus GnRH agonist in ovarian stimulation for PGD. The primary outcome measure was the number of embryos of sufficient quality for biopsy at cleavage stage. Secondary outcome parameters were the number of blastocysts available of top quality, and clinical pregnancy rate. RESULTS: There was no difference in number of oocytes retrieved, embryos at cleavage stage available for biopsy or embryo quality. The clinical pregnancy rate was higher in the GnRH agonist group; however the sample size was insufficient to allow conclusions. CONCLUSION: The use of GnRH agonist versus antagonist treatment does not result in differences in a number of oocytes, embryos or embryo quality in ovarian stimulation for preimplantation genetic diagnosis.

Serum reproductive hormone levels and ultrasound findings in female offspring after intracytoplasmic sperm injection: first results.

OBJECTIVE: To compare reproductive hormone levels and antral follicle count in intracytoplasmic sperm injection (ICSI)-conceived offspring and peers born after spontaneous conception. DESIGN: Single-center study of the reproductive health at the age of 18-22 years in the worldwide oldest cohort of female ICSI offspring. SETTING: University hospital. PATIENT(S): A longitudinally followed cohort of singleton women (n = 71) conceived by means of ICSI because of male infertility and a cross-sectionally recruited group of spontaneously conceived women of the same age (n = 81). INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Differences in serum reproductive hormone levels and ultrasound findings. RESULT(S): Median levels of antimüllerian hormone (AMH), FSH, LH, and DHEAS were similar between ICSI women and their spontaneously conceived counterparts. Median E2 levels in ICSI women taking hormonal contraceptives were higher compared with control women. A minority of ICSI women had AMH levels below the 5th or above the 95th percentile, and ICSI women were not more likely to have AMH levels below the 5th percentile or above the 95th percentile compared with control women. Mean follicle count per ovary was similar between the ICSI and control groups. Furthermore, a similar proportion of women had >19 follicles per ovary (ICSI: 20.9%; control: 20.0%). A strong positive correlation between AMH level and mean follicle count per ovary was found. CONCLUSION(S): In this cohort of 71 young adult women conceived by means of ICSI because of male infertility in their parents, antral follicle count and circulating reproductive hormone levels, including AMH, FSH, LH, and DHEAS, were found to be similar to results from peers born after spontaneous conception.

Creating a Global Community of Practice for Oncofertility.

Fertility preservation in the cancer setting, known as oncofertility, is a field that requires cross-disciplinary interaction between physicians, basic scientists, clinical researchers, ethicists, lawyers, educators, and religious leaders. Funded by the National Institutes of Health, the Oncofertility Consortium (OC) was formed to be a scientifically grounded, transparent, and altruistic resource, both intellectual and monetary, for building this new field of practice capable of addressing the unique needs of young patients with cancer. The OC has expanded its attention to include other nonmalignant conditions that can threaten fertility, and the work of the OC now extends around the globe, involving partners who together have created a community of shared effort, resources, and practices. The OC creates materials that are translated, disseminated, and amended by all participants in the field, and local programs of excellence have developed worldwide to accelerate the pace and improve the quality of oncofertility research and practice. Here we review the global oncofertility programs and the capacity building activities that strengthen these research and clinical programs, ultimately improving patient care.

Corifollitropin alfa followed by hpHMG in GnRH agonist protocols. Two prospective feasibility studies in poor ovarian responders.

In two prospective uncontrolled feasibility trials, we examined the effect of corifollitropin alfa (CFA) followed by highly purified human menopausal gonadotrophin (hpHMG) in a short flare-up gonadotropin-releasing hormone (GnRH) agonist and a long GnRH agonist protocol for women with poor ovarian response. Overall, 45 patients were treated with short flare-up and 47 patients with the long agonist protocol. All patients received a single dose of 150 µg CFA, followed by 300 IU hpHMG 7 days later, triggering with 10 000 IU hCG, CSI and day 3 embryo transfer. Ongoing pregnancy rates (OPRs) did not differ between the short 15.6% and the long 17% agonist protocol (p = 0.85). Among patients treated with the short flare-up protocol, OPRs were 20% for younger patients (<40 years old) and 12% in older women (≥40 years old), p = 0.68. Similarly, in patients treated with the long agonist protocol younger women had an OPR of 26.7% versus 12.5% in older women, p = 0.23. Among patients treated with the short flare-up, live births rate were 15% and 4.3% for younger (<40 years old) and older patients (≥40 years old), respectively, p = 0.32. Similarly, in patients treated with the long agonist protocol, live births rate were 25% and 12.9% for younger (<40 years old) and older patients (≥40 years old), respectively, p = 0.41. None of the patients reported any serious adverse event related to treatment. According to our results, CFA followed by hpHMG in a short flare-up or long GnRH agonist protocol appears to be a feasible option for poor ovarian responders. Large phase III trials are mandatory prior to introduction in clinical practice.

Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction?

Intracytoplasmic injection with testicular spermatozoa has become a routine treatment in fertility clinics. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of immature germ cells for intracytoplasmic injection has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following intracytoplasmic injection using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells to more mature stages has been proposed as a means to improve this poor outcome. Several years after the introduction of intracytoplasmic injection with elongating and round spermatids, uncertainty remains as to whether this approach can be considered a safe treatment option. This review outlines the clinical and scientific data regarding intracytoplasmic injection using immature germ cells and in vitro matured germ cells.

Intracytoplasmic spermatid injection and in vitro maturation: fact or fiction?

Intracytoplasmic injection with testicular spermatozoa has become a routine treatment in fertility clinics. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of immature germ cells for intracytoplasmic injection has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following intracytoplasmic injection using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells to more mature stages has been proposed as a means to improve this poor outcome. Several years after the introduction of intracytoplasmic injection with elongating and round spermatids, uncertainty remains as to whether this approach can be considered a safe treatment option. This review outlines the clinical and scientific data regarding intracytoplasmic injection using immature germ cells and in vitro matured germ cells.

Ovarian hyperstimulation syndrome and complications of ART.

This article reviews serious clinical complications related to assisted reproductive technology (ART) procedures (ovarian stimulation and oocyte aspiration), including ovarian hyperstimulation syndrome (OHSS), bleeding and infection. These complications are rare, but can be severe and even life-threatening. It is important that general practitioners and gynaecologists are aware of these complications, because they will often be the first to be contacted by patients. Similarly, patients should be counselled before starting ART procedures that iatrogenic complications can be associated with ovarian stimulation and/or oocyte aspiration.

ERBB-2 gene overexpression and amplification in uterine sarcomas.

BACKGROUND: The aim of this study was to determine ERBB-2 (HER-2/neu) gene alterations in different subtypes of uterine sarcomas. METHODS: After central review, representative biopsies were immunohistochemically stained and semiquantitatively scored as negative, weakly (1+), moderately (2+), or strongly (3+) positive. Subsequently, fluorescence in situ hybridization (FISH) was performed on cases with 2+ and 3+ expression. RESULTS: Seventy tumors (52 primaries and 18 recurrent) were evaluated. All 10 adenosarcomas, 21 endometrial stromal sarcomas, and 10 leiomyosarcomas were negative both in the primary and recurrent setting. Twenty-two primary carcinosarcomas were scored. The epithelial component was negative/1+ in 16 (73%), 2+/3+ in five (22.5%) tumors, and could not be evaluated in one case (4.5%), whereas the sarcoma component stained negative/1+ in 21 cases (95.5%) and 3+ (4.5%) in one case. In two recurrent carcinosarcomas, the epithelial component stained 3+ in both cases, whereas the sarcoma component scored negative and 1+. Amplification of the ERBB-2 gene as determined by FISH was observed in 3/7 (43%) carcinosarcomas with 2+ or 3+ overexpression, resulting in an overall 3/22 (14%) amplification rate. One out of four undifferentiated uterine sarcomas stained 2+. ERBB-2 immunopositivity (3+) and ERBB-2 amplification by FISH were confirmed in the recurrent tumor, resulting in a gene amplification rate of 1/4 in undifferentiated uterine sarcomas. CONCLUSION: The current results suggest absence of ERBB-2 overexpression in uterine leiomyosarcoma, uterine adenosarcoma, and endometrial stromal sarcoma, whereas the ERBB-2 gene might have a biologic role in uterine carcinosarcoma and undifferentiated uterine sarcomas.

Transition of epithelial toward mesenchymal differentiation during ovarian carcinosarcoma tumorigenesis.

BACKGROUND: It was the purpose of this study to test the monoclonal theory in ovarian carcinosarcoma. METHODS: Twenty-six women with a diagnosis of ovarian carcinosarcoma were subjected to a clinicopathologic analysis. Biopsies from metastatic lesions obtained at primary surgery and surgery for recurrent disease were reviewed. Special attention was paid to the composition of metastatic lesions and to florid desmoplastic reaction as a potential pitfall for the detection of sarcomatous areas. RESULTS: Biopsies derived from metastatic disease at primary surgery (n = 107) consisted of carcinoma cells only (n = 71, 66%), >50% carcinoma cells (n = 21, 20%), >50% sarcoma cells (n = 13, 12%), or sarcoma cells only (n = 2, 2%). The microscopic analysis demonstrated a preponderance of epithelial cells in the primary setting and suggested the epithelial component to drive the tumor, a finding consistent with the monoclonal theory. Biopsies derived from surgery for recurrent disease (n = 8) consisted of carcinoma cells only (0%), >50% carcinoma cells (n = 1, 13%), >50% sarcoma cells (n = 4, 50%), or sarcoma cells only (37%). Since sarcomatous cells dominated the tumorigenic cell population in the recurrent setting, this analysis revealed a change of the composition of metastatic lesions in time when compared to the data in the primary setting. This change was supported by the observation of a threefold higher incidence of sarcoma-dominated metastatic lesions at interval debulking when compared to primary debulking (24 vs 8%, respectively). The potential of a phenotypic change during ovarian cancer progression was further highlighted by the detection of two cases of carcinosarcoma that presented as a recurrence of epithelial ovarian carcinoma. CONCLUSION: Our results are consistent with the monoclonal theory of ovarian carcinosarcoma histogenesis, but suggest that there is a tendency toward a sarcomatous differentiation during disease progression. These data are important to understand the tumor biology and might have implications for a tailored treatment of ovarian carcinosarcoma.