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To evaluate the concentration of tumor necrosis factor α (TNF-α) and its soluble receptors (sTNFR I and II) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of TNF-α and its receptors by luteinized granulosa cells (GCs). In a cross-sectional study and through an in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI) program, 81 women undergoing oocyte retrieval were recruited. Serum and FF were obtained from 81 women. GCs were pooled from 20 patients (from six different days of oocyte retrievals, 5-16 follicles per patient). TNF-α and its soluble receptors concentration were determined by enzyme-linked immunosorbent assay and also their expression by immune cytochemistry and reverse-transcription polymerase chain reaction analysis. The median TNF-α concentration in serum was 4.06 pg/ml (interquartile range [IQR], 3.71-6.14) and significantly higher than that in FF with 3.50 pg/ml (IQR, 3.05-5.01), p < 0.001. The sTNFR I and II levels in serum were lower and higher than FF, respectively. The TNF-α levels in serum and FF of good responders were higher than low responders (p = 0.017 and 0.021, respectively). TNF-α cut-off level for low responders versus good responders was 4.174 pg/ml in serum with a pregnancy rate of 25.8% and 40% for below and above of this level, respectively (p = 0.19). For FF, the cut-off value was 3.89 pg/ml. TNF-α and its receptors were expressed by GCs. The presence of TNF-α and its soluble receptors in serum and FF and their expression by GCs suggest an important role for this cytokine in ovarian function.
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Líquido Folicular/metabolismo , Células de la Granulosa/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Estudios Transversales , Femenino , Fertilización In Vitro/métodos , Humanos , Persona de Mediana Edad , Embarazo , Inyecciones de Esperma Intracitoplasmáticas/métodos , Adulto JovenRESUMEN
STUDY QUESTION: Does preimplantation genetic testing for aneuploidy (PGT-A) by comprehensive chromosome screening (CCS) of the first and second polar body to select embryos for transfer increase the likelihood of a live birth within 1 year in advanced maternal age women aged 36-40 years planning an ICSI cycle, compared to ICSI without chromosome analysis? SUMMARY ANSWER: PGT-A by CCS in the first and second polar body to select euploid embryos for transfer does not substantially increase the live birth rate in women aged 36-40 years. WHAT IS KNOWN ALREADY: PGT-A has been used widely to select embryos for transfer in ICSI treatment, with the aim of improving treatment effectiveness. Whether PGT-A improves ICSI outcomes and is beneficial to the patients has remained controversial. STUDY DESIGN, SIZE, DURATION: This is a multinational, multicentre, pragmatic, randomized clinical trial with intention-to-treat analysis. Of 396 women enroled between June 2012 and December 2016, 205 were allocated to CCS of the first and second polar body (study group) as part of their ICSI treatment cycle and 191 were allocated to ICSI treatment without chromosome screening (control group). Block randomization was performed stratified for centre and age group. Participants and clinicians were blinded at the time of enrolment until the day after intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile couples in which the female partner was 36-40 years old and who were scheduled to undergo ICSI treatment were eligible. In those assigned to PGT-A, array comparative genomic hybridization (aCGH) analysis of the first and second polar bodies of the fertilized oocytes was performed using the 24sure array of Illumina. If in the first treatment cycle all oocytes were aneuploid, a second treatment with PB array CGH was offered. Participants in the control arm were planned for ICSI without PGT-A. Main exclusion criteria were three or more previous unsuccessful IVF or ICSI cycles, three or more clinical miscarriages, poor response or low ovarian reserve. The primary outcome was the cumulative live birth rate after fresh or frozen embryo transfer recorded over 1 year after the start of the intervention. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 205 participants in the chromosome screening group, 50 (24%) had a live birth with intervention within 1 year, compared to 45 of the 191 in the group without intervention (24%), a difference of 0.83% (95% CI: -7.60 to 9.18%). There were significantly fewer participants in the chromosome screening group with a transfer (relative risk (RR) = 0.81; 95% CI: 0.74-0.89) and fewer with a miscarriage (RR = 0.48; 95% CI: 0.26-0.90). LIMITATIONS, REASONS FOR CAUTION: The targeted sample size was not reached because of suboptimal recruitment; however, the included sample allowed a 90% power to detect the targeted increase. Cumulative outcome data were limited to 1 year. Only 11 patients out of 32 with exclusively aneuploid results underwent a second treatment cycle in the chromosome screening group. WIDER IMPLICATIONS OF THE FINDINGS: The observation that the similarity in birth rates was achieved with fewer transfers, less cryopreservation and fewer miscarriages points to a clinical benefit of PGT-A, and this form of embryo selection may, therefore, be considered to minimize the number of interventions while producing comparable outcomes. Whether these benefits outweigh drawbacks such as the cost for the patient, the higher workload for the IVF lab and the potential effect on the children born after prolonged culture and/or cryopreservation remains to be shown. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the European Society of Human Reproduction and Embryology. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. M.B.'s institution (UZBrussel) has received educational grants from IBSA, Ferring, Organon, Schering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speakers' fees from Organon, Serono Symposia and Merck. G.G. has received personal fees and non-financial support from MSD, Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, Abbott and Gedeon-Richter as well as personal fees from VitroLife, NMC Healthcare, ReprodWissen, BioSilu and ZIVA. W.V., C.S., P.M.B., V.G., G.A., M.D., T.E.G., L.G., G.Ka., G.Ko., J.L., M.C.M., M.P., A.S., M.T., K.V., J.G. and K.S. declare no conflict of interest. TRIAL REGISTRATION NUMBER: NCT01532284. TRIAL REGISTRATION DATE: 7 February 2012. DATE OF FIRST PATIENT'S ENROLMENT: 25 June 2012.
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Aneuploidia , Hibridación Genómica Comparativa/métodos , Transferencia de Embrión/estadística & datos numéricos , Cuerpos Polares , Adulto , Tasa de Natalidad , Método Doble Ciego , Transferencia de Embrión/métodos , Femenino , Humanos , Infertilidad/terapia , Análisis de Intención de Tratar , Nacimiento Vivo/epidemiología , Embarazo , Factores de Riesgo , Inyecciones de Esperma Intracitoplasmáticas/métodos , Inyecciones de Esperma Intracitoplasmáticas/estadística & datos numéricosRESUMEN
PURPOSE: Preimplantation genetic screening wants to improve artificial reproductive technologies, primarily by raising the rates of pregnancy, implantation and birth. We investigated if embryos derived from oocytes detected euploid for five chromosomes implant better than those which were biopsied but where the genetic detection failed. They were nevertheless transferred, thus serving as a sham control. METHOD: From 2004 to 2008 we performed 104 cycles of PGS with laser biopsy of the first polar body and FISH with five chromosomes. It was offered to all patients with eight or more oocytes, free of charge. The average female age was 36 years. If no euploid oocytes were available, not detected oocytes were transferred. RESULT: In 104 cycles 99 embryo transfers (95 %) were performed, resulting in 28 pregnancies (27 %), 20 births (71 %) and 8 miscarriages (29 %). The implantation rate in the euploid group was 19 vs. 13 % in the not detected group (n.s.). This trend was the same independent of age and embryo morphology. CONCLUSION: The pregnancy rate does not differ significantly from the national average. The trend in better implantation rates of euploid oocytes justifies a continuation of studies in this matter.
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Biopsia , Implantación del Embrión , Transferencia de Embrión , Pruebas Genéticas , Hibridación Fluorescente in Situ , Cuerpos Polares , Índice de Embarazo , Adulto , Aneuploidia , Femenino , Humanos , Embarazo , Diagnóstico PreimplantaciónRESUMEN
PURPOSE: For better selection of oocytes and embryos, preimplantation genetic screening (PGS) was introduced. As from the beginning of IVF, morphology was used as selection criteria; we investigated the combination of both. If there was a correlation between phenotype and genotype, invasive PGS might be replaced. METHOD: Therefore, 104 cycles with PGS were done by biopsy of the first polar body and FISH with five chromosomes. Morphology of the oocyte was recorded digitally and noted for 12 categories in 4-13 values; evaluation of the chromosomes was noted for five chromosomes in five values. Morphology and genetics were correlated to each other. RESULT: Correlations between morphology and genetics for day 0 were found: oocytes with an irregular or dark zona are less probable to have a normal chromosome 13 (80 vs. 53 %, p = 0.001). A medium amount of detritus in the perivitelline space makes it more probable to have a normal chromosome 18 (94 vs. 78 %, p = 0.001). A halo in the cytoplasm makes it less probable to be euploid for chromosome 22 (56 vs. 75 %, p = 0.018). For day 1, pattern "1, 2, 3 and fine" in the pronuclei makes it more probable to be euploid for chromosome 22 (78 vs. 63 %, p = 0.002). CONCLUSION: There are correlations between the oocyte genome and its morphology also on day 0. These correlations are not sufficient to replace PGS.
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Aneuploidia , Biopsia/métodos , Hibridación Fluorescente in Situ , Cuerpos Polares , Diagnóstico Preimplantación , Adulto , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 22 , Femenino , Humanos , Oocitos , EmbarazoRESUMEN
This study aimed to investigate whether interleukin 1ß (IL-1ß) and soluble IL-1 receptor 2 (sIL-1R2) are expressed in human granulosa cells (GCs) and relate to ovarian steroidogenesis. Ninety-six women undergoing in vitro fertilization (IVF) were recruited. RT-PCR and immunocytochemistry were used to detect mRNAs and proteins of IL-1ß and IL-1R2, respectively. The steroidogenesis of primary cultured GCs was evaluated following treatment with either IL-1ß alone or IL-1ß and FSH in combination. There were positive correlations between serum IL-1ß and serum progesterone (r = 0.220, p = 0.032) and follicular fluid (FF) estradiol (r = 0.242, p = 0.018). Additionally, serum and FF sIL-1R2 were negatively and positively correlated with FF estradiol (r = -0.376, p = 0.005) and FF progesterone (r = 0.434, p = 0.001), respectively. The mRNA and protein expression of IL-1ß and IL-1R2 became evident in GCs. IL-1ß alone significantly increased estradiol secretion from GCs, but in the presence of FSH, it could notably promote progesterone secretion in addition to estradiol. In conclusion, IL-1ß and sIL-1R2 are expressed in human GCs and substantially contribute to ovarian steroidogenesis, suggesting that the IL-1ß system may be a potential target for optimizing ovarian hyperstimulation and steroidogenesis in IVF cycles.
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Interleucina-1beta , Receptores Tipo II de Interleucina-1 , Femenino , Humanos , Células Cultivadas , Estradiol/metabolismo , Hormona Folículo Estimulante/metabolismo , Células de la Granulosa/metabolismo , Interleucina-1beta/metabolismo , Progesterona , Receptores Tipo II de Interleucina-1/metabolismoRESUMEN
Stem cell factor (SCF) plays a major role in haematopoiesis and spermatogenesis, and possibly female fertility. This study investigated the role of changes in SCF concentrations in 74 assisted conception patients. In group 1 (n=74) SCF concentration was assessed in serum and follicular fluid (FF) on the day of follicular puncture (FP) and compared in serum and FF in response to ovarian stimulation between low (n=25), moderate (n=26) and high (n=14) responders. In group 2 (n=30) serum for SCF assessment was collected throughout the menstrual cycle until gestation. SCF concentration related to the number of follicles in serum and in FF decreased from low to moderate and high responders (P<0.001); pregnancy rates were 20.0%, 34.6% and 50.1%, respectively (P=0.05). SCF in serum increased from stimulation days 6-8 to 9-11 and peaked on the day of human chorionic gonadotrophin injection (P=0.03). The SCF concentrations dropped slightly on the day of FP, increased significantly to the day of pregnancy confirmation and reached highest concentration (P=0.02) during gestation. SCF is involved in follicle development and may be a predictor of IVF outcome.
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Fertilización In Vitro , Factor de Células Madre/sangre , Biomarcadores/sangre , Gonadotropina Coriónica/farmacología , Estradiol/sangre , Femenino , Líquido Folicular/metabolismo , Humanos , Ciclo Menstrual , Folículo Ovárico/efectos de los fármacos , Inducción de la Ovulación , Embarazo , Índice de Embarazo , Factor de Células Madre/metabolismoRESUMEN
OBJECTIVE: In the context of artificial reproductive technology (ART) treatments with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), the purpose of genetic screening of oocytes and embryos in vitro prior to implantation (preimplantation genetic screening, PGS) is highly controversial. Therefore, an analysis of the following theoretical prerequisites is presented: the abstract investigation method and the medical diagnostic decision, indication and ethical acceptability. The first is a scientific task, while the other is a physician's task. THEORY OF PGS: As the new term preimplantation genetic diagnosis for aneuploidies (PGT-A) does not sufficiently take into account probable future developments, PGS is retained here. In clinical practice, PGS refers to the biopsy of polar bodies, blastomeres or trophoblast cells with indication-dependent genetic analysis. Goals include increasing pregnancy rates and reducing abortion rates, multiple birth rates, malformation rates and pointless ART treatments. To improve the pregnancy rate, PGS makes no sense if a stochastic selection advantage is not to be expected. Patients may have to choose between the chance of rapid success with a first fresh embryo transfer of blastocysts and a possibly higher overall cumulative chance of pregnancy from fresh and thawed transfers of four-to eight-cell embryos. It is neither necessary nor useful to make every medical decision dependent on randomized controlled trials (RCTs). The randomization of patients is not indicated if observational studies have not shown a positive result. For a "proof-of-principle study", the numerator and denominator of the cascade of parameters for success must be close to each other and far apart in an "efficacy study". The randomization may only be performed before the biopsy. PRACTICE: Following the introduction of blastocyst biopsy and comprehensive chromosome screening (CCS) with, for example, aCGH and NGS, referred to as "PGS 2.0â³, all RCTs since 2012 have found a positive effect. DISCUSSION: There is still disagreement about the interpretation of the results of PGS 2.0, but the overwhelming view in opinion publications seems to be that it works. This fits with the increasing global commitment to PGS 2.0. CONCLUSION: PGS may be beneficial if used with strict indications, taking into account stochastics and the will of the patient. The task of the physician, similar to counselling in prenatal medicine, is as follows: present all methods of investigation and respect the will of the patient.
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Fertilización In Vitro , Pruebas Genéticas/tendencias , Diagnóstico Preimplantación/tendencias , Aneuploidia , Blastocisto/metabolismo , Transferencia de Embrión , Femenino , Humanos , Embarazo , Inyecciones de Esperma IntracitoplasmáticasRESUMEN
OBJECTIVE: To evaluate the concentration of macrophage colony-stimulating factor (M-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of M-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF- intracytoplasmic sperm injection (ICSI) program. PATIENT(S): Serum and FF were obtained from 85 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 85 women. The GCs were pooled from 15 (3 x 5) patients (3-14 oocytes each). MAIN OUTCOME MEASURE(S): The M-CSF concentration was determined by ELISA, the expression of M-CSF and its receptor by the immunocytochemical technique and reverse transcription polymerase chain reaction analysis. In addition, M-CSF expression was investigated by cell culture time course studies. RESULTS: The median M-CSF concentration in FF (2,409.2 pg/mL) was significantly higher than that in serum (242.5 pg/mL). The M-CSF and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of M-CSF in FF than in serum and the expression of M-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.
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Fertilización In Vitro , Células de la Granulosa/fisiología , Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Transferencia de Embrión , Femenino , Líquido Folicular/metabolismo , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Luteinización/fisiología , Factor Estimulante de Colonias de Macrófagos/sangre , Embarazo , ARN Mensajero/análisis , Receptor de Factor Estimulante de Colonias de Macrófagos/sangre , Inyecciones de Esperma IntracitoplasmáticasRESUMEN
OBJECTIVE: To evaluate concentration of granulocyte colony-stimulating factor (G-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of G-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF-ICSI program. PATIENT(S): Serum and FF were obtained from 82 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 82 women. Granulosa cells were pooled from 15 patients (three experiments with five patients each; 3-14 oocytes each). MAIN OUTCOME MEASURE(S): Granulocyte colony-stimulating factor concentration was determined by ELISA, the expression of G-CSF, and its receptor by the immunocytochemical technique and reverse transcriptase polymerase chain reaction analysis. Additionally, G-CSF expression was investigated by cell culture time course studies. RESULT(S): The median G-CSF level in FF (117.98 pg/mL) was significantly higher than that in serum (67.5 pg/mL). Granulocyte colony-stimulating factor and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of G-CSF in FF than in serum and the expression of G-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.
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Líquido Folicular/metabolismo , Factor Estimulante de Colonias de Granulocitos/metabolismo , Células de la Granulosa/fisiología , Luteinización/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Adulto , Células Cultivadas , Femenino , Fertilización In Vitro , Líquido Folicular/citología , Factor Estimulante de Colonias de Granulocitos/sangre , Células de la Granulosa/metabolismo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Concentración Osmolar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inyecciones de Esperma Intracitoplasmáticas , Factores de TiempoRESUMEN
OBJECTIVE: To identify CpG sites differentially methylated in peripheral blood of men with idiopathic infertility due to impaired spermatogenesis as compared with fertile controls. DESIGN: DNA methylation profiling on peripheral blood samples using the HumanMethylation450 BeadChip (Illumina) in patients and controls, single-nucleotide polymorphism (SNP) typing by Sanger sequencing. SETTING: University institute in cooperation with genetic and infertility clinics. PATIENT(S): 30 infertile men with normal CFTR and AZF tests and karyotype, and 10 fertile male controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DNA methylation levels at CpG sites. RESULT(S): We identified 471 CpGs (287 genes) as differentially methylated between patients and controls. These were significantly enriched for the gene ontology functions MHC class II receptor activity and piwi-interacting (piRNA) binding. The latter was associated with two methylation-sensitive SNPs in the genes PIWIL1 and PIWIL2, respectively, which showed significant allele distribution skewing in the infertile cohort. We found that 445 (94.5%) of 471 differentially methylated CpGs were associated with SNPs, but 26 (15 genes) were not genomically templated, including the ENO1, MTA2, BRSK2, and LBX2 genes previously associated with fertility and spermatogenesis. CONCLUSION(S): Our study identifies surrogate DNA methylation markers for idiopathic infertility in peripheral blood and suggests that allele-specific DNA methylation differences at regulatory sites of genes involved in piRNA regulation are associated with disturbed spermatogenesis.
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Proteínas Argonautas/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Infertilidad Masculina/epidemiología , Infertilidad Masculina/genética , Espermatogénesis/genética , Adulto , Islas de CpG/genética , Metilación de ADN/genética , Frecuencia de los Genes , Marcadores Genéticos/genética , Alemania/epidemiología , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , PrevalenciaRESUMEN
INTRODUCTION: In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. METHODS: Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling) and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5). RESULTS: Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. CONCLUSION: A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures.
RESUMEN
OBJECTIVE: To evaluate the level of macrophage colony-stimulating factor (M-CSF) in serum in response to ovarian stimulation (group 1) in low-response (n = 26), moderate-response (n = 40), and high-response (n = 29) patients and to compare its changes (n = 23, group 2) throughout the menstrual cycle between pregnant and nonpregnant patients. DESIGN: Randomized controlled trial. SETTING: University IVF program. PATIENT(S): Ninety-five women undergoing IVF. INTERVENTION(S): Serum and FF collection from 95 women. MAIN OUTCOME MEASURE(S): The M-CSF concentration was determined by ELISA. RESULT(S): The M-CSF levels in FF were higher than in serum. The M-CSF levels in serum increased from low-, through moderate-, to high-response patients; pregnancy rates were 11.5%, 22.5%, and 51.7%, respectively. Levels of M-CSF in serum increased throughout stimulation until the day of oocyte retrieval and decreased until ET. During the postretrieval days, from the day of ET, through implantation, to the day of confirmation of pregnancy, the M-CSF levels of those patients who became pregnant (n = 13) increased significantly and reached their highest level. After implantation the M-CSF level decreased slightly and reached a plateau during gestation. CONCLUSION(S): Macrophage colony-stimulating factor is involved in follicle development and ovulation and could be an additional predictor for IVF outcome.
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Fármacos para la Fertilidad Femenina/uso terapéutico , Fertilización In Vitro , Hormona Folículo Estimulante Humana/uso terapéutico , Líquido Folicular/metabolismo , Infertilidad/terapia , Factor Estimulante de Colonias de Macrófagos/metabolismo , Inducción de la Ovulación , Ovulación/efectos de los fármacos , Adulto , Biomarcadores/sangre , Biomarcadores/metabolismo , Implantación del Embrión , Transferencia de Embrión , Ensayo de Inmunoadsorción Enzimática , Femenino , Edad Gestacional , Humanos , Infertilidad/sangre , Infertilidad/fisiopatología , Factor Estimulante de Colonias de Macrófagos/sangre , Masculino , Ciclo Menstrual/efectos de los fármacos , Ciclo Menstrual/metabolismo , Recuperación del Oocito , Ovulación/metabolismo , Embarazo , Índice de Embarazo , Proteínas Recombinantes/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
PROBLEM: The aim of this paper is to provide further evidence that the dystopic proliferation of endometriotic epithelia is caused by the stimulation of peritoneal macrophages. It is essential to show that endometriotic epithelial cells express the macrophage colony-stimulating factor receptor (M-CSFR) which binds the M-CSF produced by the peritoneal macrophages. METHOD OF STUDY: For the detection of M-CSFR, samples of ectopic endometrium (n = 79) and eutopic endometrium (n = 18) were compared. The specimens were gained at operative laparoscopy in the proliferative phase of the cycle. Cryostat sections were used for immunohistochemical detection. For in vitro reverse transcriptase polymerase chain reaction (RT-PCR) tests, the tissue was immediately shock frozen on paraffin sections. For the in situ RT-PCR technique the specimens were placed in a para-formaldehyde solution, embedded in paraffin and later processed. The Gene Amp 1000 in situ PCR system (Perkin Elmer) was used as the thermal cycler. RESULTS: M-CSF and the M-CSF receptor are present in eutopic and ectopic endometrium. Qualitatively, with both PCR techniques we found the M-CSF receptor to be present in all samples examined. Using the histochemical detection technique, the M-CSF receptor was found in nearly 70% of endometriosis patients compared with a statistically significant lower percentage in normal endometrium. CONCLUSIONS: The in situ RT-PCR technique and immunohistochemistry elaborated the need to trace the cellular sources of the M-CSF receptor. The identification of the M-CSF receptor in endometriotic tissue and in endometrium is apt to open a new experimental field in endometriosis research.