Isolation of human mesenchymal stem cells from amnion, chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols.

OBJECTIVE: To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue. RESULTS: A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol. CONCLUSIONS: The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.

The experience of pregnancy resulting from Assisted Reproductive Technology (ART) treatment: A qualitative Brazilian study.

BACKGROUND: Pregnancies achieved through medical treatments following a period of infertility may demand extra emotional and practical investment from women. AIM: This paper aims at understanding the experience of pregnancy after Assisted Reproductive Technology, and exploring whether this experience is affected by previous failed infertility treatments. METHODS: This paper uses a qualitative approach. Participants were nineteen expectant first-time mothers from Brazil who conceived through Assisted Reproductive Technology treatment. During the third trimester of gestation, a semi-structured interview was administered to assess perceptions of and feelings about treatment and pregnancy. Interview transcripts were analysed using thematic analysis, and the sample was divided into two groups according to whether it was the participant's first treatment or not. FINDINGS: Themes identified include: tolerance of the demands of treatment and pregnancy, consideration of the mechanics of treatment and pregnancy, and emotionally painful aspects of treatment and pregnancy. Pregnancy itself was regarded as a reward or compensation for the difficulties undergone. Perspectives differed according to whether pregnancy followed the first Assisted Reproductive Technology treatment; those who had undergone previously unsuccessful treatments focused less on the mechanical aspects of the process but were more concerned about possible physical problems. CONCLUSION: The similarities and differences found according to number of treatments attempted should be taken into consideration when providing psychological support for expectant Assisted Reproductive Technology mothers.

What variables predict generic and health-related quality of life in a sample of Brazilian women experiencing infertility?

BACKGROUND: Infertility is a condition associated with impairment in several areas of life. Questionnaires about quality of life (QoL) allow the examination of the impact of health conditions in a broader way, comprehending outcomes beyond symptomatology, morbidity and mortality. The aim of this study was to identify factors associated with various aspects of QoL. METHODS: Cross-sectional study using the following: a socio-demographic and clinical data form, the Health Survey Short Form (SF-36) which examines health-related QoL and the WHOQOL-BREF which examines general QoL. RESULTS: 177 women seeking fertility assistance were interviewed. The sample was predominantly composed of women between 30 and 40 years old (64%), who had known about their infertility for <5 years (57%) and who had had no previous attempts at assisted reproduction (79%). Logistic regression indicated the following predictor variables: age (for better general health and physical functioning), previous in vitro fertilization (for lower vitality and poor psychological health scores), previous reproductive tract surgery (for worse general health but higher environment scores), advanced education (for higher vitality, mental health and environment scores, but for worse social relationships) and perception of worse sexual life (for lower overall scores). CONCLUSIONS: The identification of factors associated with better or worse QoL, in its different domains, is vital in order to propose and test scientifically based interventions on infertile women.

Sperm recovery prediction in azoospermic patients using Doppler ultrasonography.

PURPOSE: To evaluate power Doppler ultrasonography to predict sperm recovery in azoospermic patients. METHODS: Color Doppler and power Doppler ultrasonography of testis were performed in 38 patients before testicular sperm extraction. Analysis of blood flow included the pulsatility and resistance index of intratesticular vessels and testicular artery, and power Doppler of testis. The results of power Doppler of testis were classified into three categories: 0, no vessels found; 1, one to three vessels; 2, more than three vessels found. RESULTS: Power Doppler of both testis showed a significant difference between obstructive azoospermia and non-obstructive azoospermia (Fisher's exact test - P = 0.02), and between the groups with and without sperm recovery (Fisher's exact test - P = 0.001). Doppler indices of intratesticular vessels and testicular artery were similar between the groups. CONCLUSIONS: Testicular Power Doppler assessment showed that patients with obstructive azoospermia have better blood flow than patients with non-obstructive azoospermia, and power Doppler is able to predict sperm recovery in azoospermic patients.

Subendometrial power Doppler quantification: a new classification proposal.

Fifty-two women with regular menses were enrolled in the study. The patients were not allowed to use non-steroidal anti-inflammatory drugs within 24 h of any examination. All patients were examined during the mid-luteal phase (6-9 days after ovulation, according to previous ultrasound record). Power Doppler energy levels were classified into five categories according to the per cent area of sub-endometrial signal: I (<10%), II (10-25%), III (25-50%), IV (50-75%) and V (>75%). The colour Doppler signal was considered positive when it reached at least the endometrial basal layer. The picture of the endometrium was analysed and the regions of interest were identified and marked for further analysis. Each recorded image was then independently evaluated and classified by three blinded observers. According to the power Doppler classification, age, body mass index (BMI) and endometrial thickness were analysed, and no significant differences were observed among them. The Kappa test (0.70) demonstrated an excellent agreement among examiners (P = 0.0001). This study has validated a very simple and cost-effective classification for sub-endometrial vascularization. This method of quantification may potentially be of use, and its relevance to clinical practice should be explored.

Insulin-like growth factor-1 and insulin-like growth factor binding protein-1 and 3 in the follicular fluid of infertile patients submitted to in vitro fertilization.

PURPOSE: In the present article we propose to evaluate IGF-1, IGFBP-1 and 3 in the follicular fluid of infertile patients submitted to in vitro fertilization. METHODS: We performed a case-control study with 53 infertile patients submitted to the first in vitro fertilization attempt. We compared their follicular fluid concentration of IGF-1, IGFBP-1 and IGFBP-3 between the patients who became pregnant (n = 11) versus those nonpregnant (n = 42). RESULTS: The clinical characteristics of patients from the two groups were similar in terms of age and body mass index. Data related to the analysis of ovulation induction was not different regarding length of induction in days, number of retrieved oocytes, fertilization rate, and number of transferred embryos. Furthermore, the number of FSH units required for ovarian induction was also similar between the studied groups. IGF-1 and IGFBP-1 were not significantly different between the groups (p > 0.05). However, those patients that became pregnant presented a lower follicular fluid concentration of IGFBP-3, 2237.10 +/- 582.73 pg/ml and 2657.64 +/- 584.15 ng/ml, respectively (p = 0.038). CONCLUSIONS: We demonstrated an association of a lower follicular fluid IGFBP-3 in individuals that became pregnant compared to subjects that did not after in vitro fertilization.

Predictive factors for motile sperm recovery using testicular biopsy in nonobstructive azoospermic patients.

OBJECTIVE: The objective of the present study was to assess the predictive value of hormone values, histological analysis for the finding of motile spermatozoa on testicular biopsy in nonobstructive azoospermia. METHODS: This cross-sectional study assessed serum follicle-stimulating hormone (FSH), luteinizing hormone, prolactin and total testosterone values in 50 patients undergoing testicular biopsy prior to ICSI. P < 0.05 was considered significant. RESULTS: Recovery was successful in 28 cases, and motile spermatozoa were retrieved in 7. Significant differences were detected between presence of motile spermatozoa and absence of spermatozoa in terms of FSH values (P = 0.003, one-way ANOVA). The other variables did not present statistical differences. A receiver operating characteristic curve showed that FSH levels below 17.00 IU/L were predictive of motile spermatozoa recovery. CONCLUSIONS: FSH values can serve as a predictive factor for the recovery of motile spermatozoa using biopsy in azoospermic patients. On the other hand, histological analysis and other hormone values were not helpful in the prediction of motile sperm.

Insulin-like growth factor (IGF)-1 and IGF binding protein-1 and -3 in the follicular fluid of infertile patients with endometriosis.

BACKGROUND: Endometriosis is associated with pituitary-ovarian axis dysfunction. The study of the follicular fluid in patients with endometriosis is important to elucidate the pathophysiological mechanism of this disease. The objective of this present paper was to analyse the dosages of insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 and 3 (IGFBP-1 and IGFBP-3) in the follicular fluid environment of infertile patients with endometriosis. METHODS: A total of 41 infertile patients undergoing IVF between January 1999 and January 2000 participated in the cross-sectional prospective study. Patients were divided into three groups: group I, minimal/mild endometriosis (n = 12); group II, moderate/severe endometriosis (n = 10); and group III, tubal obstruction (n = 19). The ultra-short protocol was used in association with recombinant FSH for ovulation induction. Follicular fluid analysis was performed using radioimmunoassay with specific kits. RESULTS: Follicular fluid IGF-1 and IGFBP-3 levels were not significantly different among the groups; however, follicular fluid IGFBP-1 levels were lower in those patients with moderate/severe endometriosis (P < 0.05). Comparison of ovulation induction time, number of recombinant FSH units, number of follicles, oocytes and embryos, and fertilization and gestation/cycle rates showed non-significant differences. CONCLUSION: Infertile patients with moderate/severe endometriosis, which is associated with ovulatory dysfunction, presented lower levels of IGFBP-1 in the follicular fluid when undergoing IVF.

Prolactin and growth hormone secretion after thyrotrophin-releasing hormone infusion and dopaminergic (DA2) blockade in infertile patients with minimal/mild endometriosis.

BACKGROUND: The origin of infertility in patients with endometriosis without tubal occlusion has not yet been clearly defined. Several reports show an abnormal pituitary-ovarian axis in this group of patients. Moreover, prolactin (PRL) and growth hormone (GH) secretion is closely related to reproductive status. This study aimed to evaluate PRL and GH secretion after metoclopramide and thyrotrophin-releasing hormone (TRH) infusion in infertile patients with minimal/mild endometriosis. METHODS: A total of 64 women participated in the study: 33 fertile patients without endometriosis; 10 fertile patients with minimal/mild endometriosis; and 21 infertile patients with minimal/mild endometriosis. TRH or metoclopramide was administered randomly in two sequential menstrual cycles (cycle days 3-5). Serum PRL and GH secretion before and after dopaminergic type 2 (DA2) receptor blockade and TRH were compared. RESULTS: Higher serum PRL levels were observed in patients with endometriosis at baseline and after 15 and 30 min of TRH administration. Also, infertile patients with endometriosis had lower serum estradiol levels than fertile patients. Moreover, the dopaminergic blockade did not result in abnormal PRL or GH secretion. CONCLUSIONS: Decreased serum estradiol levels and altered PRL secretion after TRH administration in infertile patients with minimal/mild endometriosis are related to ovulatory dysfunction and infertility in this group of patients without tubal occlusion.

Growth hormone and prolactin secretion after metoclopramide administration (DA2 receptor blockade) in fertile women.

In this report, we will describe the results of a cross-sectional study to assess PRL and GH secretion during the early follicular phase in 22 fertile patients after metoclopramide administration in order to achieve a dopaminergic DA2 receptor blockade. Blood samples were collected at - 15, 0, 15, 30, 45 and 60 minutes. PRL, GH, estradiol, IGF-I, TSH, glucose, and insulin were measured in the samples taken at - 15 and 0 minutes. The existence of a correlation between GH and PRL secretion was investigated. All patients presented normal serum levels of estradiol, prolactin, insulin, fasting glucose and IGF-I. Serum GH levels were not changed after metoclopramide infusion (p = 0.302), but there was a significant alteration in serum PRL (p = 0.0001) with the highest levels after 30 (mean: 237.20 ng/ml +/- 95.86) and 45 (mean: 211.80 ng/ml +/- 83.24) minutes. Serum GH levels did not correlate with serum PRL levels after the dopaminergic DA2 blockade. We conclude that GH secretion was not modulated by a direct effect of type 2 dopamine receptor.

Hyperprolactinemia and luteal insufficiency in infertile patients with mild and minimal endometriosis.

The objective of the present paper was to assess the presence of hormonal alterations in infertile women with stage I or II endometriosis (Group III, n = 20) compared to fertile women without endometriosis (Group I, n = 14) and to fertile women with endometriosis (Group II, n = 7). Serum levels of FSH, LH, estradiol, TSH, and PRL were measured between days 1 and 5 of the early follicular phase; in the luteal phase, three serum samples were collected for progesterone measurement, and endometrial biopsies were performed. Serum estradiol levels were lower (p = 0.035) in infertile patients with endometriosis than in fertile patients without endometriosis. Six infertile patients with endometriosis presented prolactin levels above 20 ng/ml. This was not observed in the other groups. Luteal insufficiency was more frequent in infertile patients with endometriosis (78.9%) than in fertile patients with (42.9%) or without endometriosis (0%). In a multiple logistic regression analysis, only the presence of endometriosis and infertility was significantly associated with luteal insufficiency. The serum levels of LH, FSH, and TSH were not significantly different among the groups. Luteal insufficiency and altered prolactin secretion were associated with endometriosis, and could be important mechanisms causing infertility in this group of patients.

Histerossonossalpingografia e histerossalpingografia no diagnóstico de permeabilidade tubária em pacientes inférteis / Histerosonosalpingography and Histerosalpingography in the Diagnosis of tubal patency in infertility patients

OBJETIVOS: Comparar a histerossonossalpingografia (HSS) em relaçäo à histerossalpingografia (HSG) na avaliaçäo de fator tubário em pacientes inférteis. MATERIAL E MÉTODO: Foi realizado um estudo transversal com 30 pacientes em investigaçäo de infertilidade, com idade inferior a 38 anos. As pacientes foram submetidas à HSS, HSG e videolaparoscopia (LPC) na primeira fase do ciclo menstrual. Foi comparada a avaliaçäo de permeabilidade tubária da HSS e da HSG, utilizando-se a LPC como padräo-ouro. Foi calculado o valor preditivo negativo (VP-) dos exames. A HSS utilizou como meio de contraste o Ecovist®, a HSG utilizou contraste iodado hidrossolúvel e a LPC utilizou a cromotubagem com azul de metileno. Foi considerada como diferença estatisticamente significativa um p<0,05. RESULTADOS: A amostra final de estudo foi de 26 pacientes (quatro abandonaram a investigaçäo). A idade média da amostra foi de 30,6 anos. A HSS demonstrou permeabilidade tubária em pelo menos uma das tubas em 24 pacientes (92,3 por cento). A HSG demonstrou permeabilidade em uma das tubas em 25 (96,2 por cento), e a LPC em 25 pacientes (96,2 por cento). O VP- da HSS foi de 92 por cento e o VP- da HSG foi de 100 por cento. Näo houve diferença estatística significante entre a HSS e a HSG (p=0,996, teste de Yates). CONCLUSÕES: A HSS é um método alternativo à HSG na avaliaçäo de permeabilidade tubária de pacientes inférteis

[Hysterosonosalpingography and hysterosalpingography in the diagnosis of tubal patency in infertility patients]. / Histerossonossalpingografia e histerossalpingografia no diagnóstico de permeabilidade tubária em pacientes inférteis.

OBJECTIVES: To compare the hysterosonosalpingography(HSS) to the hysterosalpingography(HSG) in the evaluation of the tubal factor in infertility patients. MATERIAL AND METHODS: A transversal study with 30 patients in infertility investigation, with age minor than 38 years was performed. The patients were submitted to HSS, HSG and laparoscopy (LPC) in the first cycle menstrual phase. The evaluation of tubal patency of HSS and HSG, with LPC like gold-standard was compared. It was calculated the negative predictive value (PV-) of the exams. The HSS used Ecovist like contrast agent, the HSG used water soluble contrast media and the LPC used tubal insufflation with methylene blue. It was considered significative a p< 0.05 RESULTS: The final sample was of 26 patients (4 leave the investigation). The mean age was of 30,6 years. The HSS showed tubal patency in at least one of the tubes in 24 patients (92,3%). The HSG showed tubal patency in one of the tubes in 25 patients (96,2%), and the LPC in 25 patients (96,2%). The PV- of the HSS was of 92% and the PV- of HSG was of 100%. The differences were not statistically significant (p=0.996, Yates test). CONCLUSIONS: The HSS is an alternative method in the evaluation of tubal patency in infertility patients.

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein.

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control. NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40 percent by PF from controls (PFc) and 50 percent by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60 percent) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control, NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11 percent) and PFe/PFi groups (17 percent), and the ionophore-induced AR was higher for PFi (33 percent) than PFc/PFe (25 percent). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR

Glycosidases in the peritoneal fluid from infertile women with and without endometriosis.

OBJECTIVES: To investigate the activity and biochemical properties of glycosidases in the peritoneal fluid of infertile patients with or without endometriosis in comparison with fertile women. DESIGN AND METHODS: Peritoneal fluid was collected from 29 women undergoing a laparoscopy. The sample was separated into the following groups: fertile women (n = 11), infertile with endometriosis (n = 11), and infertile without endometriosis (n = 7). The activity of glycosidases was determined with specific p-nitrophenylglycosides as colorimetric substrates. RESULTS: The activity of alpha-fucosidase, alpha-glucosidase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, and beta-N-acetylhexosaminidase was investigated. Enzymatic activities of alpha-fucosidase and beta-N-acetylglucosaminidase were detected in higher amounts than other glycosidases. The activities of alpha-fucosidase and N-acetylglucosaminidase were increased in the case of infertile patients without endometriosis, while beta-galactosidase was increased in endometriosis patients. Enzyme properties as pH optimum, pH stability, thermal stability and inhibition by specific carbohydrates were similar for both control and infertile samples. Analysis of kinetic parameters indicate that Vmax values of glycosidases were higher for infertile samples than their respective controls. CONCLUSIONS: The results indicate that higher amounts of glycosidases are present in the peritoneal fluid from infertile patients. The elevated activity of these hydrolytic enzymes suggest possible deleterious effect on gametes, and could explain some cases of infertility.