Fertility preservation after gonadotoxic treatments for cancer and autoimmune diseases.

BACKGROUND: The indications for fertility preservation (FP) have expanded. A few patients who underwent gonadotoxic treatment did not have the opportunity to receive FP, leading to concerns that these patients may develop premature ovarian insufficiency. However, the usefulness of FP in women with reduced ovarian reserve has also been questioned. Progestin-primed ovarian stimulation can improve the controlled ovarian stimulation (COS) protocol, but there is limited data on the efficacy of FP with progestin-primed ovarian stimulation. METHODS: We conducted a prospective study of 43 women with cancer or autoimmune diseases before and after gonadotoxic treatment at the reproductive unit of Keio University Hospital, counselled between 1 January 2018 and 31 December 2021. After counselling, informed consent was obtained for FP from 43 patients, with those who underwent gonadotoxic treatment of the primary disease being prioritised. Gonadotropin-releasing hormone analogue or progestin was used to suppress luteinising hormone in COS before or after gonadotoxic treatment. The number of cryopreserved mature oocytes was the primary outcome. RESULTS: Forty-three patients and 67 assisted reproductive technology cycles were included in the analysis. The median age at entry was 32 [inter quartile range (IQR), 29-37] years. All patients in the post-gonadotoxic treatment group had their oocytes frozen. Gonadotoxic treatment resulted in fewer oocytes [median 3 (IQR 1-4); pre-gonadotoxic treatment group: five patients, 13 cycles] vs. median 9 (IQR 5-14; pre-gonadotoxic treatment group: 38 patients, 54 cycles; P < 0.001). Although anti-Müllerian hormone levels were lower in the post-gonadotoxic treatment group (n = 5, 13 cycles, median 0.29 (IQR 0.15-1.04) pg/mL) than in the pre-gonadotoxic treatment group (n = 38, 54 cycles, median 1.89 (IQR 1.15-4.08) pg/mL) (P = 0.004), oocyte maturation rates were higher in the post-gonadotoxic treatment group [median 100 (IQR 77.5-100) %] than in the pre-gonadotoxic group [median 90.3 (IQR 75.0-100) %; P = 0.039]. Five patients in the pre-gonadotoxic treatment group had their cryopreserved embryos thawed, of which three had live births. CONCLUSIONS: Oocytes obtained for FP from women with cancer or autoimmune disease for FP are of satisfactory quality, regardless of whether they are obtained post-gonadotoxic treatment or COS protocols.

Embryonic ß-Catenin Is Required for Priming of the Uterus to Implantation.

Repeated implantation failure is a major cause of infertility among healthy women. Uterine ß-catenin (CTNNB1) plays a critical role in implantation. However, the role of embryonic CTNNB1 during implantation remains unclear. We addressed this topic by analyzing mice carrying Ctnnb1-deficient (Ctnnb1Δ/Δ) embryos. Ctnnb1Δ/Δ embryos were produced by intercrossing mice bearing Ctnnb1-deficient eggs and sperms. We found that Ctnnb1Δ/Δ embryos developed to the blastocyst stage; thereafter, they were resorbed, leaving empty decidual capsules. Moreover, leukemia inhibitory factor, a uterine factor essential for implantation, was undetectable in Ctnnb1Δ/Δ blastocysts. Furthermore, CDX2, a transcription factor that determines the fate of trophectoderm cells, was not observed in Ctnnb1Δ/Δ blastocysts. Intrauterine injection with uterine fluids (from control mice) and recombinant mouse leukemia inhibitory factor proteins rescued the uterine response to Ctnnb1Δ/Δ blastocysts. These results suggest that embryonic CTNNB1 is required for the secretion of blastocyst-derived factor(s) that open the implantation window, indicating that the uterine response to implantation can be induced using supplemental materials. Therefore, our results may contribute to the discovery of a similar mechanism in humans, leading to a better understanding of the pathogenesis of repeated implantation failure.

Impact of the COVID-19 pandemic on surgery for benign diseases in gynecology: A nationwide survey by the Japan Society of Obstetrics and Gynecology.

AIM: This study aimed to clarify the impact of coronavirus disease 2019 on gynecology practice in Japan, in particular, on surgeries for benign gynecological diseases. METHODS: An online questionnaire was distributed to 966 facilities in Japan, including core facilities, facilities participating in perinatal and gynecologic oncology registries, and facilities certified for training by the Japanese Society of Obstetrics and Gynecology Endoscopy. The number of surgeries performed was compared between 2019 and 2020, when the novel coronavirus disease was prevalent. RESULTS: Five hundred and eighty (58.2%) facilities responded. The total number of surgeries decreased from 129 648 in 2019 to 118 565 in 2020, by 8.5%, for all surgical procedures. However, there was a clear increase in the number of robotic surgeries performed in 2020 compared to that in 2019 for all populations. The number of total hysterectomies decreased markedly from 15 385 in 2019 to 12 531 in 2020, a fall of 10.1%. CONCLUSIONS: The number of surgeries for benign gynecological diseases decreased by 8.5% in 2020 compared to that in 2019. This value is among the lowest in the world.

The possible effects of the Japan Society of Clinical Oncology Clinical Practice Guidelines 2017 on the practice of fertility preservation in female cancer patients in Japan.

Purpose: In 2017, the first guidelines for fertility preservation in cancer patients were published in Japan. However, the impact of the guidelines remains unknown. Therefore, the authors conducted a nationwide survey on cryopreservation procedures in the period from shortly before to after publication of the guidelines (2016-2019) and compared the results with our previous survey (2011-2015). The authors also surveyed reproductive specialists' awareness of the guidelines and implementation problems. Methods: The authors sent a questionnaire to 618 assisted reproductive technology facilities certified by the Japanese Society of Obstetrics and Gynecology. Results: The authors received responses from 395 institutions (63.8%). Among them, 144 institutions conducted cryopreservation for cancer patients (vs. 126 in 2011-2015) and performed 2537 embryo or oocyte and 178 ovarian tissue cryopreservation procedures (vs. 1085 and 122, respectively). Compared with the previous period, indications were more varied and protocols for controlled ovarian stimulation were more standardized. Reproductive specialists' interest in oncofertility was high, but many reported three main difficulties: selecting a treatment method, storing samples in the long term, and securing the necessary human resources. Conclusions: The practice of fertility preservation in cancer patients in Japan has been considerably affected by the first Japanese guidelines.

Zscan5b Deficiency Impairs DNA Damage Response and Causes Chromosomal Aberrations during Mitosis.

Zygotic genome activation (ZGA) begins after fertilization and is essential for establishing pluripotency and genome stability. However, it is unclear how ZGA genes prevent mitotic errors. Here we show that knockout of the ZGA gene Zscan5b, which encodes a SCAN domain with C2H2 zinc fingers, causes a high incidence of chromosomal abnormalities in embryonic stem cells (ESCs), and leads to the development of early-stage cancers. After irradiation, Zscan5b-deficient ESCs displayed significantly increased levels of γ-H2AX despite increased expression of the DNA repair genes Rad51l3 and Bard. Re-expression of Zscan5b reduced γ-H2AX content, implying a role for Zscan5b in DNA damage repair processes. A co-immunoprecipitation analysis showed that Zscan5b bound to the linker histone H1, suggesting that Zscan5b may protect chromosomal architecture. Our report demonstrates that the ZGA gene Zscan5b is involved in genomic integrity and acts to promote DNA damage repair and regulate chromatin dynamics during mitosis.

Membrane protein CD9 is repositioned and released to enhance uterine function.

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice.

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Clinical efficacy of a combination of Percoll continuous density gradient and swim-up techniques for semen processing in HIV-1 serodiscordant couples.

To evaluate the clinical efficacy of a procedure comprising a combination of Percoll continuous density gradient and modified swim-up techniques for the removal of human immunodeficiency virus type 1 (HIV-1) from the semen of HIV-1 infected males, a total of 129 couples with an HIV-1 positive male partner and an HIV-1 negative female partner (serodiscordant couples) who were treated at Keio University Hospital between January 2002 and April 2012 were examined. A total of 183 ejaculates from 129 HIV-1 infected males were processed. After swim-up, we successfully collected motile sperms at a recovery rate as high as 100.0% in cases of normozoospermia (126/126 ejaculates), oligozoospermia (6/6), and asthenozoospermia (36/36). The recovery rate of oligoasthenozoospermia was 86.7% (13/15). In processed semen only four ejaculates (4/181:2.2%) showed viral nucleotide sequences consistent with those in the blood of the infected males. After using these sperms, no horizontal infections of the female patients and no vertical infections of the newborns were observed. Furthermore, no obvious adverse effects were observed in the offspring. This protocol allowed us to collect HIV-1 negative motile sperms at a high rate, even in male factor cases. We concluded that our protocol is clinically effective both for decreasing HIV-1 infections and for yielding a healthy child.

Factors affecting pregnancy outcomes in young women treated with fertility-preserving therapy for well-differentiated endometrial cancer or atypical endometrial hyperplasia.

BACKGROUND: Patients hoping to preserve their fertility receive conservative treatment with high-dose medroxyprogesterone acetate (MPA) for well-differentiated endometrioid adenocarcinoma (EC) or atypical endometrial hyperplasia (AEH) . Such treatment generally involves frequent intrauterine operations, including dilation and curettage (D&C) and endometrial biopsy (EMB), which could result in endometritis, endometrial thinning, or intrauterine adhesion. In turn, any of these outcomes could adversely affect implantation and pregnancy development. The current study thus aimed to identify factors that might affect pregnancy following conservative treatment by MPA. METHODS: We compared a pregnancy group (45 patients) with a non-pregnancy group (53 patients) of MPA-treated patients to evaluate the factors affecting clinical pregnancy establishment. We undertook a multivariate logistic regression analysis based on factors shown by univariate analysis to be significantly different between the groups. Univariate analysis identified number of D&C, endometrial thickness, duration of MPA administration, age of pregnancy permission (the age at which a patient was first allowed to attempt pregnancy after disappearance of the lesion), period of disappearance of lesions, and recurrence as independent variables. RESULTS: The odds ratios (95 % confidence interval) of multivariate analysis for disease recurrence, endometrial thickness during ovulation, and age of pregnancy permission were 0.283 (0.102-0.785), 1.677 (1.251-2.248), and 0.889 (0.792-0.998), respectively. There was no significant difference in the other independent variables between groups. CONCLUSIONS: We identified three factors considered to affect pregnancy establishment following conservative treatment with MPA: recurrence, endometrial thickness during ovulation, and the age of the pregnancy permission. Introduction of infertility treatment including assisted reproductive technology (ART) soon after achieving tumor disappearance by MPA would therefore be beneficial for patients with disease recurrence, thin endometrium, or a higher age of pregnancy permission.

Pregnancy Outcomes After Abdominal Radical Trachelectomy for Early-Stage Cervical Cancer: A 13-Year Experience in a Single Tertiary-Care Center.

OBJECTIVE: To investigate pregnancy outcomes in women after abdominal radical trachelectomy (RT) for early-stage cervical cancer. METHODS: The patients' background, fertility, and pregnancy outcomes were reviewed in a total of 61 pregnancies in 48 of 172 women who underwent abdominal RT at Keio University Hospital between September 2002 and December 2013. RESULTS: There were 5 women with stage IA1, 2 with stage IA2, and 41 with stage IB1. Histological types were as follows: squamous cell carcinoma (n = 36), adenocarcinoma (n = 10), and adenosquamous cell carcinoma (n = 2). The pregnancy rate of women attempting to conceive after abdominal RT was 44% (48/109). The mean ± SD duration from abdominal RT to conception was 3.1 ± 1.9 years. Of 61 pregnancies, 42 pregnancies were achieved by fertility treatment (in vitro fertilization-embryo transfer, 39; intrauterine insemination, 3). After excluding one pregnancy without detailed clinical information, there were 42 live births (5 in 22-27 weeks, 11 in 28-33weeks, 20 in 34-36 weeks, and 6 in 37-38 weeks), 13 miscarriages, and 5 ongoing pregnancies. While there were 10 first trimester miscarriages, 3 pregnancies ended in the second trimester owing to chorioamnionitis. The mean gestational age at birth was 33 weeks of pregnancy. Thirty-seven neonates were appropriate-for-date, and one was small-for-date. Six pregnancies exhibited massive bleeding from the residual cervix in the late pregnancy. Preterm birth less than 34 weeks of pregnancy was related to premature rupture of the membrane (P < 0.05). Chorioamnionitis was evident in 9 of 11 pregnancies with preterm premature rupture of the membrane followed by birth at less than 34 weeks of pregnancy. No parturients exhibited lochiometra and endometritis postpartum. CONCLUSIONS: Abdominal RT provided favorable pregnancy outcomes, and fertility treatment could be advantageous to conception. Massive bleeding from the residual cervix as well as ascending infection might be characteristic features during pregnancy after abdominal RT.

Infertility etiologies are genetically and clinically linked with other diseases in single meta-diseases.

The present review aims to ascertain whether different infertility etiologies share particular genes and/or molecular pathways with other pathologies and are associated with distinct and particular risks of later-life morbidity and mortality. In order to reach this aim, we use two different sources of information: (1) a public web server named DiseaseConnect ( http://disease-connect.org ) focused on the analysis of common genes and molecular mechanisms shared by diseases by integrating comprehensive omics and literature data; and (2) a literature search directed to find clinical comorbid relationships of infertility etiologies with only those diseases appearing after infertility is manifested. This literature search is performed because DiseaseConnect web server does not discriminate between pathologies emerging before, concomitantly or after infertility is manifested. Data show that different infertility etiologies not only share particular genes and/or molecular pathways with other pathologies but they have distinct clinical relationships with other diseases appearing after infertility is manifested. In particular, (1) testicular and high-grade prostate cancer in male infertility; (2) non-fatal stroke and endometrial cancer, and likely non-fatal coronary heart disease and ovarian cancer in polycystic ovary syndrome; (3) osteoporosis, psychosexual dysfunction, mood disorders and dementia in premature ovarian failure; (4) breast and ovarian cancer in carriers of BRCA1/2 mutations in diminished ovarian reserve; (5) clear cell and endometrioid histologic subtypes of invasive ovarian cancer, and likely low-grade serous invasive ovarian cancer, melanoma and non-Hodgkin lymphoma in endometriosis; and (6) endometrial and ovarian cancer in idiopathic infertility. The present data endorse the principle that the occurrence of a disease (in our case infertility) is non-random in the population and suggest that different infertility etiologies are genetically and clinically linked with other diseases in single meta-diseases. This finding opens new insights for clinicians and reproductive biologists to treat infertility problems using a phenomic approach instead of considering infertility as an isolated and exclusive disease of the reproductive system/hypothalamic-pituitary-gonadal axis. In agreement with a previous validation analysis of the utility of DiseaseConnect web server, the present study does not show a univocal correspondence between common gene expression and clinical comorbid relationship. Further work is needed to untangle the potential genetic, epigenetic and phenotypic relationships that may be present among different infertility etiologies, morbid conditions and physical/cognitive traits.

Derivation of human decidua-like cells from amnion and menstrual blood.

We induced differentiation of human amnion-derived mesenchymal stem cells (AMCs) and menstrual blood-derived mesenchymal stem cells (MMCs) into endometrial stroma-like cells, which could be useful for cell therapy to support embryo implantation. Interestingly, the expression patterns of surface markers were similar among AMCs, MMCs, and endometrial stromal cells. In addition, whereas treatment with estrogen and progesterone was not very effective for decidualizing AMCs and MMCs, treatment with 8-Br-cAMP prompted remarkable morphological changes in these cells as well as increased expression of decidualization markers (prolactin and insulin-like growth factor binding protein-1) and attenuated expression of surface markers unique to mesenchymal stem cells. These results demonstrated that bone marrow-derived stem cells, which are considered a potential source of endometrial progenitor cells, as well as AMCs and MMCs show in vitro decidualization potential, which is characteristic of endometrial stromal cells.

Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition.

In mammals, uterine epithelium is remodeled cyclically throughout adult life for pregnancy. Despite the expression of CD9 in the uterine epithelium, its role in maternal reproduction is unclear. Here, we addressed this issue by examining uterine secretions collected from patients undergoing fertility treatment and fertilization-competent Cd9(-/-) mice expressing CD9-GFP in their eggs (Cd9(-/-)TG). CD9 in uterine secretions was observed as extracellular matrix-like feature, and its amount of the secretions associated with repeated pregnancy failures. We also found that the litter size of Cd9(-/-)TG female mice was significantly reduced after their first birth. Severely delayed re-epithelialization of the endometrium was then occurred. Concomitantly, vascular endothelial growth factor (VEGF) was remarkably reduced in the uterine secretions of Cd9(-/-)TG female mice. These results provide the first evidence that CD9-mediated VEGF secretion plays a role in re-epithelialization of the uterus.

ß-catenin functions pleiotropically in differentiation and tumorigenesis in mouse embryo-derived stem cells.

The canonical Wnt/ß-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. ß-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if ß-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established ß-catenin-deficient (ß-cat(Δ/Δ)) mouse embryo-derived stem cells and showed that ß-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs). However, teratomas formed from embryo-derived ß-cat(Δ/Δ) ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional ß-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that ß-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight ß-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

Reproductive and obstetric outcomes after radical abdominal trachelectomy for early-stage cervical cancer in a series of 31 pregnancies.

STUDY QUESTION: What are the reproductive and obstetric outcomes in patients undergoing radical abdominal trachelectomy (RAT) for early-stage cervical cancer? SUMMARY ANSWER: When RAT was performed before a pregnancy achieved with fertility treatments, pregnancy rate of 36.2% was obtained and 71.4% of these women gave birth at ≥ 32 weeks of gestation. WHAT IS KNOWN ALREADY: Reproductive and obstetric outcomes after radical vaginal trachelectomy (RVT) are well documented; however, these outcomes after RAT have not been well studied. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study of patients at a single institution who underwent RAT and became pregnant. Reproductive and obstetric outcomes of 114 patients who had undergone RAT from September 2002 to December 2010 were investigated. PARTICIPANTS/MATERIAL, SETTING, METHODS: Women of reproductive age with early-stage cervical cancer who wished to preserve their fertility were documented. MAIN RESULTS AND THE ROLE OF CHANCE: Patients' median age was 33 years (25-40 years). A total of 31 pregnancies were achieved in 25 patients and 6 patients had 2 pregnancies. Eighteen of 25 patients (72.0%) had infertility problems; 17 patients conceived with IVF-embryo transfer and 1 patient with intrauterine insemination. The pregnancy rate among patients who wished to conceive was 36.2% (25/69). Among 31 pregnancies in 25 patients, 4 patients had first trimester miscarriage and 1 patient had second trimester miscarriage. Excluding the five patients who miscarried and the five ongoing pregnancies, all the 21 patients had deliveries by Cesarean section. Four patients had a preterm birth in the second trimester and 17 patients delivered in the third trimester. Of the 17 pregnancies that reached the third trimester, 2 (11.8%) were preterm births between 29 and 32 weeks, 11 (64.7%) were delivered between 32 and 37 weeks and 4 (23.5%) at ≥ 37 weeks of gestation. LIMITATIONS, REASONS FOR CAUTION: Because of the retrospective data collection, not all pregnancies may have been recorded. WIDER IMPLICATIONS OF THE FINDINGS: Prospective multicenter studies are needed to determine if the results shown in this retrospective cohort can be generalized to all patients with early-stage cervical cancer who wish to undergo the fertility-sparing RAT procedure.

Buoyant density and sedimentation dynamics of HIV-1 in two density-gradient media for semen processing.

OBJECTIVE: To compare buoyant density and sedimentation kinetics of human immunodeficiency virus 1 (HIV-1) in two sperm-washing media, Percoll and Pureception. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): None. INTERVENTION(S): Buoyant density and sedimentation kinetics of HIV-1 particles (MOLT-4/LAI strain) were measured in Percoll and Pureception using isopyknic ultracentrifugation and continuous-density-gradient centrifugation. MAIN OUTCOME MEASURE(S): The HIV-1 particles were detected and semiquantified using a reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 RNA. RESULT(S): Calculated buoyant density of HIV-1 was approximately 1.042 in both media in isopyknic centrifugation. However, most HIV-1 particles were found in fractions with specific gravity less than 1.04 in both media, even after 40 minutes of density-gradient centrifugation at 1,600 g. Small viral accumulations were observed at the bottom of the tube in Pureception density gradients. CONCLUSION(S): Although we found very high efficiency of HIV-1 removal using density-gradient centrifugation, a minute quantity of virus was found at the bottom of the gradient tube when Pureception was used as the medium.

Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation.

Delayed implantation (embryonic diapause) occurs when the embryo at the blastocyst stage achieves a state of suspended animation. During this period, blastocyst growth is very slow, with minimal or no cell division. Nearly 100 mammals in seven different orders undergo delayed implantation, but the underlying molecular mechanisms that direct this process remain largely unknown. In mice, ovariectomy before preimplantation ovarian estrogen secretion on day 4 of pregnancy initiates blastocyst dormancy, which normally lasts for 1-2 weeks by continued progesterone treatment, although blastocyst survival decreases with time. An estrogen injection rapidly activates blastocysts and initiates their implantation in the progesterone-primed uterus. Using this model, here we show that among approximately 20,000 genes examined, only 229 are differentially expressed between dormant and activated blastocysts. The major functional categories of altered genes include the cell cycle, cell signaling, and energy metabolic pathways, particularly highlighting the importance of heparin-binding epidermal growth factor-like signaling in blastocyst-uterine crosstalk in implantation. The results provide evidence that the two different physiological states of the blastocyst, dormancy and activation, are molecularly distinguishable in a global perspective and underscore the importance of specific molecular pathways in these processes. This study has identified candidate genes that provide a scope for in-depth analysis of their functions and an opportunity for examining their relevance to blastocyst dormancy and activation in numerous other species for which microarray analysis is not available or possible due to very limited availability of blastocysts.

Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells.

Decidual growth factors and locally produced cytokines are thought to activate specific phosphorylation signalling pathway(s), thereby eliciting a variety of decidual functions. We have previously reported the activation of c-Src tyrosine kinase during ovarian steroid-induced decidualization of cultured human endometrial stromal cells. As chicken c-Src is known to be activated upon dephosphorylation of tyrosine 527 (Y527, corresponding to Y530 in human), we here employed a monoclonal antibody, clone 28, directed against the active form of human c-Src whose Y530 is dephosphorylated, and investigated whether c-Src became dephosphorylated at Y530 and thereby activated during decidualization. We found that the active form of c-Src was up-regulated and demonstrated increased kinase activity during in-vitro decidualization. Immunohistochemistry revealed that decidual cells in early pregnancy decidua were intensely stained with clone 28 when compared with the stromal cells in the non-pregnant endometrium. Moreover, the active form of c-Src translocated from a perinuclear region to the cytoplasm upon decidualization. Thus, the Y530 dephosphorylation, kinase activation, and subcellular translocation of c-Src may be intracellular signalling events associated with decidualization in vivo as well as in vitro.