Human endometrial stromal cells inhibit the pro-angiogenic stimulus of hCG in vitro.

During embryo implantation, endometrial angiogenesis is regulated by signals originating from the endometrium itself and the developing embryo. It has been suggested that hCG may play a pro-angiogenic role; therefore, we sought to understand its regulatory role in blood vessel formation in human endometrium using in vivo and in vitro models. In the in vivo model, we screened 16 angiogenesis-related transcripts in the endometrium upon intrauterine administration of hCG. Oocyte donors were recruited and during their controlled ovarian stimulation cycle received a single dose of hCG or vehicle on the day of oocyte pick up during a cycle of ovarian stimulation. One hour before obtaining an endometrial sample, women received an intrauterine administration of vehicle or hCG (500, 1500 and 5000 IU). Transcript and protein analysis showed that MMP3 and VEGFA increased, whereas TIMP1 decreased. The in vitro analysis studied the angiogenic potential of conditioned medium (CM) from primary cultures of human endometrial stromal cells (ESC) stimulated with hCG. Using a 2D and 3D in vitro angiogenesis assays, our results indicate that CM from ESC almost completely inhibits the capillary-like structure formation in endothelial cells, overriding the pro-angiogenic effect of hCG; and this inhibition due to secreted factors present in CM specifically reduced the migration potential of endothelial cells. In conclusion, the endometrial stromal milieu seems to modulate the direct pro-angiogenic effects of hCG on endothelial cells during embryo implantation.

Research priorities in infertility and assisted reproductive technology treatments - a James Lind Alliance priority setting partnership with brazilian patients.

OBJECTIVE: To identify the main research interests of Brazilian patients in the field of infertility and assisted reproductive technology (ART) treatments. METHODS: This prospective multicenter cross-sectional study was carried out in Brazil. Patients attending five fertility centers from the Huntington Group between October and December 2018 were invited to join the study, which consisted of answering an anonymous survey online. Two hundred and twenty-seven patients signed the informed consent form and were emailed the survey link. The survey was designed based on the James Lind Alliance Priority Setting Partnership protocol. In the area of infertility, patients were probed on issues such as somatic and psychological effects of treatment, prevention, assisted reproductive technology (medications and procedures), success rates, risks, and emotional aspects. RESULTS: The response rate (RR) was 47.58% (108 patients; 88 women - RR 51.46% and 20 men - RR 35.71%). Patient mean age was 36.5 years (SD 4.6). The top ten research priorities listed were 1) short- and long-term side effects of treatment; 2) how to cope with infertility; 3) risks associated with ART; 4) success rates in ART; 5) impact of diet on ART and fertility; 6) healthy habits; 7) alternative therapies; 8) impact of exercise on fertility and ART success; 9) oocyte quality and ovarian reserve; and 10) genetic or inherited causes of infertility. CONCLUSION: To better cater to the needs of patients and develop patient-centered care in the field of infertility and ART treatment, clinicians, healthcare providers, and the scientific community must identify patient concerns and priorities and make efforts to address them.

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo.

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1-2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo's missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.

The relationship among HOXA10, estrogen receptor α, progesterone receptor, and progesterone receptor B proteins in rectosigmoid endometriosis: a tissue microarray study.

BACKGROUND: Very few studies have evaluated the expression of homeobox A10 (HOXA10) and steroid (estrogen and progesterone) receptors exclusively in deep endometriosis. Conclusions drawn from studies evaluating peritoneal and ovarian endometriosis are usually generalized to explain the pathogenesis of the disease as a whole. We aimed to evaluate the expression of HOXA10, estrogen receptor α (ER-α), progesterone receptor (PR), and PR-B in rectosigmoid endometriosis (RE), a typical model of deep disease. METHODS: We used RE samples from 18 consecutive patients to construct tissue microarray blocks. Nine patients each were operated during the proliferative and secretory phases of the menstrual cycle. We quantified the expressions of proteins by immunohistochemistry using the modified Allred score. RESULT: The HOXA10 was expressed in the stroma of nodules during the secretory phase in 5 of the 18 patients. Expression of ER-α (in 16 of 18 patients), PR (in 17 of 18 patients), and PR-B (17 of 18 patients) was moderate to strong in the glands and stroma of nodules during both phases. Expression of both PR (P = .023) and PR-B (P = .024) was significantly greater during the secretory phase. CONCLUSION: The HOXA10 is expressed in RE, where it likely imparts the de novo identity of endometriotic lesions. The ER-α, PR, and PR-B are strongly expressed in RE, which differs from previous studies investigating peritoneal and ovarian lesions. This suggests different routes of pathogenesis for each of the 3 types of endometriosis.

Evaluation of FAS and caspase-3 in the endometrial tissue of patients with idiopathic infertility and recurrent pregnancy loss.

OBJECTIVE: To investigate expression of BcL-2, FAS, FAS ligand (FASL) and cleaved caspase-3 in the endometrial tissue of women with idiopathic infertility (with two consecutive failed cycles of in vitro fertilization) and women with idiopathic recurrent pregnancy loss. The control group consisted of fertile women. STUDY DESIGN: Endometrial tissue samples from fertile women (n=25), women with idiopathic infertility (n=25) and women with idiopathic recurrent pregnancy loss (n=25) were collected on the seventh or eighth postovulatory day of their menstrual cycles for evaluation. Expression of BcL-2, FAS, FASL and cleaved caspase-3 was assessed using immunohistochemical methods. RESULTS: Expression of BcL-2 and FAS was significantly higher and lower, respectively, in the women with idiopathic infertility than in the other groups (p<0.01). Expression of cleaved caspase-3 was significantly lower in the women with idiopathic infertility than in the other groups (p<0.01). Expression of FASL was similar in all three groups. CONCLUSION: Disturbances in endometrial apoptosis may be a contributing factor in patients with idiopathic infertility and recurrent pregnancy loss.

Laboratory methods in the study of endometrial Claudin-4.

Immunohistochemistry is a suitable method for the detection of proteins from the Claudin family and several antibodies are commercially available for the detection of Claudin congeners. Immunodetection of Caludin-4 in the paraffin-embedded specimens might be a useful tool for studying the role of these proteins in the cyclic transformation of the endometrium and its role in the endometrial receptivity; furthermore, other components of the junctional zone involved in the transformational process of the endometrium can be detected by means of immunohistochemistry/immunofluorescence with several polyclonal or monoclonal antibodies. The aim of this chapter is to comprehensively overview the materials and methods to perform the endometrial biopsy and to detect Claudin-4 in paraffin-embedded samples of endometrium. Additionally, the interpretation of the results is addressed.

State of the art of assisted human reproduction

Background: Infertility is a disease observed in approximately 10% of the reproductive age population (20-44 years old), and is defined as the failure to conceive after twelve months of regular sexual intercourse, without contraception; in women older than 35 years old, this period is reduced to 6 months. The main causes of infertility are tubal, ovarian and uterine and sperm abnormalities, endometriosis, and those with undetermined causes. Over the past 30 years, several techniques were developed to overcome these factors including gamete cryopreservation, controlled ovarian stimulation, intra-uterine insemination, in vitro fertilization, intracytoplasmatic sperm injection). Review: Despite advances in assisted reproductive technologies (ART), treatment success is still strongly dependent on oocyte and sperm quality, and resulting embryo viability. The most promising advance on oocyte quality assessment is the evaluation of the ovarian reserve by the quantification of the anti-müllerian hormone (AMH). Since ovarian reserve is closely related to oocyte quality, AMH levels could be an indicator of both oocyte production capacity and the potential of these oocytes to generate a viable embryo. On the other hand, despite the development of techniques to overcome male factor infertility, attention has been paid on the semen evaluation, since routine sperm evaluation techniques are known to be ineffective, especially in those cases of unexplained infertility. Therefore, techniques were developed to assess acrosome and membrane integrity, mitochondrial potential, DNA integrity, and fertilizing capacity of sperm. However, further studies are necessary to evaluate sperm DNA integrity without damaging the cell, allowing the injection of a spermatozoon with an intact DNA when using ICSI. Regarding embryo quality, even with a good quality oocyte (as assessed by the current techniques) and an apparently normal sperm, there are still chances of generating an embryo with genetic abnormalities. In such cases, and in cases of recurrent failures, women over 35 years of age, and couples with a pre-existing genetic risk, the preimplantation genetic diagnosis (PGD) appears to be an important tool to improve the odds of pregnancy and avoid abortions or the conception of fetuses with genetic abnormalities. The technique of PGD, usually performed with PCR or FISH, has gained a powerful tool with the development of the Comparative Genomic Hybridization (CGH). However, recent studies aiming to identify markers of oocyte and sperm quality and embryo viability are in course using mass spectroscopy. With this sensitive technique applied to body fluids (i.e., blood, follicular fluid, seminal plasma), granulosa cells, sperm, and culture media, researches are being conducted to non-invasively identify biomarkers that will help understand reproductive mechanisms and to efficiently predict the outcome of ARTs. Conclusion: Significant advances in ART have been observed in the last few years, yet, failures still occur with high frequency. This review will focus on techniques to assess oocyte quality, sperm function and embryo viability, aiming to provide tools for a precise prognosis when treating infertile couples.

Successful interval laparoscopic transabdominal cervicoisthmic cerclage preceding twin gestation: a case report.

Laparoscopic transabdominal cervicoisthmic cerclage (LTCC) is an alternative, less-morbid option to the traditional transabdominal cerclage, indicated for patients with cervical incompetence. Experience with the technique is based on case reports and a few case series. Considering LTCC for twin gestations, reports are very scarce and are derived from LTCC performed during pregnancy. We report the case of a 36-year-old patient, gravida 1, para 0, aborta 1, who underwent interval LTCC after a previous failed transvaginal emergency cerclage performed in the second trimester. Hysteroscopic metroplasty was concomitantly performed for an incomplete septate uterus. The procedure lasted 100 minutes, with an estimated blood loss of 50 mL. The patient was discharged home on the second postoperative day. The patient became pregnant with twins 3 months after the procedure after undergoing in vitro fertilization. The gestational course was uneventful, and the patient delivered 2 healthy neonates at 38 weeks gestation by elective cesarean section. The cerclage tape was left in situ. Minor modifications of the previously reported techniques included use of a laparoscopic Deschamps needle for placing the cotton cardiac tape used as suture material. Vessels in the cervical transverse cervical ligament were exposed before cerclage tape application. To our knowledge, this is the first report of interval LTCC preceding a twin gestation.

Should the gynecologist perform laparoscopic bowel resection to treat endometriosis? Results over 7 years in 168 patients.

STUDY OBJECTIVE: To assess the feasibility and safety of laparoscopic bowel resections for endometriosis performed by gynecologic surgeons. DESIGN: Retrospective cohort study (Canadian Task Force Classification II-3). SETTING: Fertility and pelvic surgery clinics. PATIENTS: One hundred sixty-eight women (age 21-53 years) with symptoms including pelvic pain, infertility, or both with 252 bowel endometriotic lesions underwent laparoscopic bowel resection performed by gynecologic surgeons between May 2000 and January 2008. INTERVENTIONS: Laparoscopic procedures for excision of several endometriotic nodes and lesions included shaving resection (LscShR), discoid resection (LscDR), segmental resection (LscSgR), terminal ileal resection (LscIR), partial cecal resection (LscCR), and appendectomy (LscAp). MEASUREMENTS AND MAIN RESULTS: The 168 patients underwent 172 laparoscopic bowel resections (4 patients were operated on twice) by the same surgeon. Lesions were distributed as follows: 133 (79%) in the rectum, 61 (24%) in the sigmoid colon, 47 (19%) in the appendix, 5 (2%) in the terminal ileum, 3 (1%) in the descending colon, and 3 (1%) in the cecum. At surgeon discretion, 12 lesions were not resected. A total of 216 bowel procedures were necessary to remove the 240 lesions include shaving resection in 22 patients (10%), discoid resection in 52 (24%), segmental resection in 92 (42%), terminal ileal resection in 2 (1%), partial cecal resection in 1 (0.6), and appendectomy in 47 (22%). Major complications occurred in 13 patients (7.6%) and included rectovaginal fistula in 3 patients (1.7%), rectosigmoid anastomosis dehiscence and bowel occlusion in 1 patient each (0.6%), and persistent bowel dysfunction in 4 patients (2.3%). These results are comparable to those reported in the literature to date. Complete relief of symptoms (measured using the Visick scale) was noted in patients with dysmenorrhea (59%), dyspareunia (75%), noncyclic pelvic pain (90%), pain on defecation (100%), constipation (83%), and cyclic rectal bleeding (100%). CONCLUSION: Surgery to treat bowel endometriosis can be safely and efficiently performed by the gynecologic pelvic surgeon. Meticulous training and a multidisciplinary approach to comprehensive operative care are necessary. These findings can be validated by prospective collaborative studies and reports from other surgeons.

Células natural killer endometriais: o que são? O que fazem? O que devemos saber? / Endometrial natural killer cells: what are they? What do they do? What do we need to know?

As células natural killer endometriais, também chamadas células natural killer uterinas, têm recebido especial atenção no campo da imunologia reprodutiva. Teorias que consideram alterações na resposta imune como uma causa de infertilidade conjugal e de falhas nos tratamentos de reprodução assistida têm ponderado um possível envolvimento negativo das células natural killer endometriais. As células natural killer são linfócitos que podem ser identificados no sangue periférico e no endométrio, apresentando diferenças fenotípicas e funcionais importantes. As células periféricas não se alteram com a fase do ciclo menstrual e implantação, sendo que as células natural killer endometriais apresentam variações durante o ciclo menstrual e período peri-implantacional, com menores concentrações durante a fase proliferativa e aumentando na segunda fase do ciclo. A célula natural killer endometriais participam nas várias fases da implantação, invasão trofoblástica, placentação e desenvolvimento fetal e no desenvolvimento da gestação humana até aproximadamente 20 semanas.

Endometrial natural killer cells have been given special attention in reproductive immunology. The relation between the endometrial natural killer cells and alterations in the immune response as a cause of couples infertility and failure in assisted reproduction treatment have been studied in several theories. Natural killer cells are lymphocytes that may be identified in peripheral blood and endometrium, with phenotypical and functional differences between them. Peripheral natural killer cells do not change with the menstrual cycle or implantation, as opposed to endometrial natural killer cells which present lower concentration in the proliferative phase and higher concentration in the luteal phase. Endometrial natural killer cells play an important role in the implantation, trophoblastic invasion, placentation, fetal development and development of the human pregnancies up to 20 weeks of gestation.

Protein profile of the luteal phase endometrium by tissue microarray assessment.

To investigate the luteal phase endometrial expression of leukemia inhibitor factor (LIF), insulin-like growth factor 1 (IGF-1), progesterone receptor (PR), claudin 4 (CLDN4), vascular-endothelial growth factor receptor 3 (VEGFR-3), bone morphogenetic protein 4 (BMP-4) and citokeratin 7 (CK-7), we obtained luteal phase endometrial samples from 52 women. Samples were dated and integrated using a tissue microarray (TMA). Samples were immunostained for LIF, IGF-1, PR, CLDN4, VEGFR-3, BMP-4 and CK-7. Frequencies of positive expressions at the early, mid and late luteal phases were compared by two proportions test. Concomitant expression of these proteins was assessed with Chi-square or Fischer's test. The frequency of LIF was positively correlated to the frequency of IGF-1 (r = 0.99; p < 0.05) and PR (r = 0.99; p < 0.05), and the correlation between IGF-1 and PR tended to be significant (r = 0.98; p < 0.1). The expression of PR was associated with the absence of CLDN4 (p < 0.001). Thus, expression of LIF, IGF-1 and PR are correlated during the luteal phase, and immunohistochemistry for these proteins might be used to assist in the assessment of endometrial maturation. In addition, the expression of CLDN4 and PR was not concomitant, warranting further investigation on the relationship of their endometrial expression.

Effect of GnRH down-regulation on cumulus cell viability and apoptosis as measured by fluorescence-activated cell sorting.

OBJECTIVE: To determine whether gonadotropin releasing hormone (GnRH)-agonist or -antagonist induces higher percentages of cumulus cell apoptosis and if the use of either is detrimental to ART outcomes. PATIENTS: Women in a private facility under treatment for IVF had their cumulus cells isolated and analyzed by flow cytometry. Viable, apoptotic, and dead cumulus cell rates related to ovarian stimulation by GnRH-agonist or -antagonist were measured and compared with fertilization and implantation rates. RESULTS: Treatment with GnRH-agonist produced a greater number of follicles than treatment with GnRH-antagonist. No differences in implantation and pregnancy rates were found. While cumulus cell (CC) apoptosis was positively correlated with estradiol on the day of hCG administration, no significant difference in the percentage of apoptotic cells between treatments was detectable. Additionally, implantation rate and the average follicular estradiol production on the day of hCG administration were no different between treatments. CONCLUSIONS: GnRH-agonist or -antagonist treatment protocols induce similar levels of apoptosis in CCs and are not detrimental to ART outcomes.

Transplante de tecido ovariano / Ovarian tissue transplant

O congelamento de tecido ovariano para ser transplantado é um procedimento que pode reservar a função reprodutiva e endócrina desse órgão no futuro. Constitui uma alternativa para pacientes jovens com câncer e submetidas a quimioterapia, sem prole constituída, que desejam engravidar. Embora a lesão celular e as modificações moleculares possam ocorrer em decorrência do processo de congelamento e de descongelamento, há relatos de transplantes bem sucedidos tanto em animais quanto em seres humanos. A técnica de congelamento pode ter aplicações nos programas de Reprodução Humana, além de poder ser útil para evitar eventuais repercussões do hipoestrogenismo em mulheres que foram ooforectomizadas precocemente devido ao tratamento de neoplasias.

Metoclopramide-induced hyperprolactinaemia caused marked decline in pinopodes and pregnancy rates in mice.

BACKGROUND: The impact of hyperprolactinaemia on endometrial function, along with embryo implantation, has been the subject of discussion. This article examines whether experimental hyperprolactinaemia can affect mouse ovarian function, endometrial pinopodes and embryo implantation. METHODS: For pinopode analysis, 60 female mice were randomly divided into two groups: control (vehicle) and experimental [metoclopramide (MCP) 200 microg per day]. Injections were given subcutaneously for 50 days, and then, normally cycling females were housed with male mice for copulation during proestrus. The animals were killed on the fifth day following coitus when the antimesometrium portions of the uterine horns were removed for endometrial analysis. Blood was collected for prolactin (PRL) determination. In the second experiment, 60 female mice were used to evaluate the ovarian function by measuring estrogen and progesterone levels and counting luteal bodies and oocytes in the oviduct and uterus during estrus. RESULTS: The highest pregnancy rates and the largest population of pinopodes were both found in the vehicle group (P<0.01). Estrogen and progesterone levels in MCP-treated mice were lower than those in control mice (P<0.05). Also, the number of implantations was significantly lower in the MCP-treated group compared with the vehicle group after embryo transfer (P<0.001). CONCLUSION: PRL seems to have suppressive effects on ovarian function and the number of pinopodes; conceivably, hyperprolactinaemia has a negative effect on mouse embryo implantation.

The treatment of infertility and its historical context.

The objective of this review is to analyse and integrate into a semi-historical background the alternative treatments available for the alleviation of infertility. Data are presented on their causes and success, using compilations of data reported in the literature along with personal experience. Results are expressed as the outcome of endocrinological and surgical treatments for female and male infertility. Clinical pregnancy and delivery rates are reported as the measurements of effectiveness of in-vitro fertilization and related techniques. Detailed analysis of the most important considerations with respect to a number of infertility treatment options are provided, and show how the treatment of choice will always depend on the specific context and needs of each patient. New technologies are described briefly, and an ethical comment completes the review.