Cristiano Oliveira

Ultrastructure of tubular smooth endoplasmic reticulum aggregates in human metaphase II oocytes and clinical implications.

OBJECTIVE To compare demographic, embryologic, pregnancy, and newborn outcomes after intracytoplasmic sperm injection (ICSI) cycles with or without mature oocytes (metaphase II [MII]) showing visible aggregates of tubular smooth endoplasmic reticulum (aSERT) and to describe the ultrastructure of this dysmorphism. DESIGN Retrospective study. SETTING Private fertility center and university cell biology and genetics departments. PATIENT(S) There were 721 ICSI cycles, 520 carrying morphologically normal MII (control group) and 60 containing aSERT-MII (study group). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Embryologic and clinical and live birth outcomes, including malformations and ultrastructural characterization of aSERT-MII. RESULT(S) Compared with the control group there was a significant decrease in the fertilization, embryo cleavage, and blastocyst rates in the study group. The only child born after transfer of embryos derived from aSERT-MII presented a major cardiovascular malformation. Ultrastructurally, large aSERT were surrounded by abnormal-shaped mitochondria and clusters of small dense bodies formed by very small vesicles, and they had curvilinear dense tubules in the interior. The same pathology was observed in small peripheral aSERT. CONCLUSION(S) The presence of large aSERT, showing attainment of the periphery, demonstrated that the cytoplasm is pathologic. The compromised embryo development and implantation was associated with decreased clinical outcomes and newborn malformations. Therefore, oocytes with large aSERT should not be used for embryo transfer.

Treatment by testicular sperm extraction and intracytoplasmic sperm injection of 65 azoospermic patients with non‐mosaic Klinefelter syndrome with birth of 17 healthy children

The aim of this work was to present the clinical and embryological outcomes of 65 azoospermic patients with non‐mosaic Klinefelter syndrome (KS), treated by testicular sperm extraction (TESE), followed by intracytoplasmic sperm injection (ICSI), either with fresh or cryopreserved testicular spermatozoa. In total, spermatozoa were recovered in 25/65 (38.5%) of the cases. Of the 48 patients who choose to perform TESE followed by ICSI using fresh testicular spermatozoa (treatment TESE), spermatozoa was recovered in 19 patients (40%), with birth of 12 newborn. Of the 17 patients who choose to perform TESE followed by testicular sperm cryopreservation, spermatozoa were recovered in six patients (35%), with birth of one child. Of the patients who performed treatment TESE, nine went for a new cycle using cryopreserved spermatozoa. Of these, five patients had a previous failed treatment cycle (two patients, three newborn) and four with a previous success went for a new cycle (one patient, one newborn). Overall, the embryological and clinical rates were as follows: 52% of fertilization, 41% of blastocyst, 27% of implantation, 39% of live birth delivery and 47% of newborn. Of the 16 clinical pregnancies, 14 had a successful delivery (12 girls and 5 boys). The 17 newborns had a mean gestation time of 37.2 weeks (35.3% pre‐term) and a mean newborn weight of 2781.3 g (37.5% low weight). Comparisons between cycles with fresh and frozen‐thaw spermatozoa revealed higher fertilization and clinical pregnancy rates with fresh spermatozoa, with no differences regarding implantation or newborn rates. Of the 17 newborns, no abnormal karyotypes (n = 3) or numerical abnormalities in chromosomes 13, 18, 21, X and Y (n = 14) as evaluated by Multiplex Ligation–dependent Probe Amplification were observed. In conclusion, this study presents further data that reassures that men with KS have no increased risk of transmitting their genetic problem to the offspring.

Pregnancy and birth after intracytoplasmic sperm injection with totally immotile sperm recovered from the ejaculate

OBJECTIVE To report the birth of two healthy children after intracytoplasmic sperm injection (ICSI) with totally immotile spermatozoa recovered from the ejaculate. DESIGN Retrospective case report. SETTING University-based hospital. PATIENT(S) Four couples in whom spermatozoa recovered from the ejaculate were totally immotile but presented normal vitality scores. INTERVENTION(S) Therapeutical IVF-ET attempts coupled with ICSI. MAIN OUTCOME MEASURE(S) Fertilization and pregnancy results after ICSI. RESULTS With random sperm injection, 19 of the 36 injected oocytes showed normal fertilization and cleavage. One of four patients had a twin pregnancy that resulted in birth of two healthy children. CONCLUSION(S) In cases in which totally immotile ejaculated sperm present normal vitality scores, normal clinical outcomes can be achieved by using the usual random sperm selection during conventional ICSI.

Urinary estrogen metabolites and self-reported infertility in women infected with Schistosoma haematobium

Background Schistosomiasis is a neglected tropical disease, endemic in 76 countries, that afflicts more than 240 million people. The impact of schistosomiasis on infertility may be underestimated according to recent literature. Extracts of Schistosoma haematobium include estrogen-like metabolites termed catechol-estrogens that down regulate estrogen receptors alpha and beta in estrogen responsive cells. In addition, schistosome derived catechol-estrogens induce genotoxicity that result in estrogen-DNA adducts. These catechol estrogens and the catechol-estrogen-DNA adducts can be isolated from sera of people infected with S. haematobium. The aim of this study was to study infertility in females infected with S. haematobium and its association with the presence of schistosome-derived catechol-estrogens. Methodology/Principal Findings A cross-sectional study was undertaken of female residents of a region in Bengo province, Angola, endemic for schistosomiasis haematobia. Ninety-three women and girls, aged from two (parents interviewed) to 94 years were interviewed on present and previous urinary, urogenital and gynecological symptoms and complaints. Urine was collected from the participants for egg-based parasitological assessment of schistosome infection, and for liquid chromatography diode array detection electron spray ionization mass spectrometry (LC/UV-DAD/ESI-MSn) to investigate estrogen metabolites in the urine. Novel estrogen-like metabolites, potentially of schistosome origin, were detected in the urine of participants who were positive for eggs of S. haematobium, but not detected in urines negative for S. haematobium eggs. The catechol-estrogens/ DNA adducts were significantly associated with schistosomiasis (OR 3.35; 95% CI 2.32–4.84; P≤0.001). In addition, presence of these metabolites was positively associated with infertility (OR 4.33; 95% CI 1.13–16.70; P≤0.05). Conclusions/Significance Estrogen metabolites occur widely in diverse metabolic pathways. In view of the statistically significant association between catechol-estrogens/ DNA adducts and self-reported infertility, we propose that an estrogen-DNA adduct mediated pathway in S. haematobium-induced ovarian hormonal deregulation could be involved. In addition, the catechol-estrogens/ DNA adducts described here represent potential biomarkers for schistosomiasis haematobia.

Impact of GnRH ovarian stimulation protocols on intracytoplasmic sperm injection outcomes

BackgroundAlthough a large number of studies have been conducted in relation to ovarian response and pregnancy after GnRH agonist and GnRH antagonist controlled ovarian hyperstimulation protocols, most of them used single or combinations of a few predictive factors, and none included the stimulation protocol in the multivariable analysis. The present study was thus primarily designed to investigate the predictive value of the stimulation protocol and to analyze the possible relationships between stimulation protocols and treatment outcomes after adjusting for a large set of variables that potentially affect reproductive outcomes. Factors related to pregnancy achievement and predictive of the number of oocytes retrieved and high quality of the embryos obtained were also analyzed.MethodsTo analyze the impact of GnRH ovarian stimulation protocols on the independent predictors of ovarian response, high quality embryos and clinical pregnancy, two groups out of 278 ICSI treatment cycles were compared prospectively, 123 with a GnRH agonist and 155 with a GnRH antagonist, with multivariable analysis assessing outcomes after adjusting for a large set of variables.ResultsAntagonists were significantly associated with lower length and total dose of GnRH, lower length of rFSH, and higher numbers of oocytes and high quality embryos, whereas the agonist presented a higher fertilization rate and probability of pregnancy. Significant predictors of retrieved oocytes and high quality embryos were the antagonist protocol, lower female age, lower serum levels of basal FSH and higher total number of antral follicles. Significant predictors of clinical pregnancy were the agonist protocol, reduced number of attempts, increased endometrial thickness and lower female age. The probability of pregnancy increased until 30 years-old, with a decline after that age and with a sharp decline after 40 years-old.ConclusionThe models found suggest that not only the protocol but also factors as female age, basal FSH, antral follicles, number of attempts and endometrial thickness should be analyzed for counselling patients undergoing an ICSI treatment.

Immunohystochemical analysis of CFTR in normal and disrupted spermatogenesis

Cystic fibrosis is the most frequent autosomal recessive disease in the Caucasian population, with an incidence of 1:2500 newborn and a frequency of 1:25. The associated gene is Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and it encodes the CFTR protein that functions as a chloride (Cl−) channel. It is found in the apical membrane of exocrine epithelial cells, responsible for the regulation of the movement of water and solutes through biological membranes. To our knowledge, there are no studies on protein localization in the different cell types of the seminiferous epithelium with different pathologies. The aim of the present study was to analyze the expression of the CFTR protein in the human seminiferous epithelium of infertile males with different pathologies. CFTR protein expression was studied by immunohistochemistry in paraffin sections of testicular biopsies of six infertile men: Sertoli cell only syndrome, maturation arrest, secondary obstructive azoospermia, primary obstructive azoospermia due to congenital bilateral absence of the vas deferens (CBAVD), severe oligozoospermia, and retrograde ejaculation. All cell types of the seminiferous epithelium were studied: Sertoli cells, spermatogonia, primary spermatocytes at the leptotene/zygotene and at the pachytene stages, secondary spermatocytes, round, elongating and elongated spermatids, and spermatozoa. With the exception of sperm, all cells were labeled in the cytoplasm and in the cytoplasmic membrane. In the patient with CBAVD labeling was light at the cell membrane and absent in the cytoplasm of Sertoli cells and diploid germ cells. Generally, labeling was stronger after the diploid stage, which is probably related to cell volume reduction during spermiogenesis. The results obtained also suggest that the CFTR protein may impact CBAVD spermatogenesis and other pathologies.

Corresponding Ganglion Cell Atrophy in Patients With Postgeniculate Homonymous Visual Field Loss.

Background: The goal of our study was to look for the presence of homonymous ganglion cell layer–inner plexiform layer complex (GCL-IPL) thinning using spectral-domain optical coherence tomography (SD-OCT) in patients with a history of adult-onset injury to the postgeniculate pathways with rigorous radiological exclusion of geniculate and pregeniculate pathology. Methods: We performed a retrospective review of twenty-two patients (ages 24–75 y, 6 men, 16 women) with homonymous visual field (VF) defects secondary to postgeniculate injury examining the GCL-IPL with SD-OCT. An additional fifteen patients (ages 28–85 y, 5 men, 10 women) with no visual pathway pathology served as controls. Using segmentation analysis software applied to the macular scan, a normalized asymmetry score was calculated for each eye comparing GCL-IPL thickness ipsilateral vs contralateral to the patients brain lesions. Results: We found that 15 of the twenty-two subjects had a relative thinning of the GCL-IPL ipsilateral to the postgeniculate lesion in both eyes (represented by a positive normalized asymmetry score in both eyes), whereas a similar pattern of right/left asymmetry was found in 4 controls (P = 0.0498). The magnitude of asymmetry was much greater in subjects compared with controls (P = 0.0004). There was no association between the degree of GCL-IPL thinning and the mean deviation on automated VF testing. A moderate correlation (R = 0.782, P = 0.004) between the magnitude of thinning and latency from onset of retrogeniculate injury was observed only after excluding patients beyond a cutoff point of 150 months. Conclusions: This data provides compelling new evidence of retrograde transsynaptic degeneration causing retinal ganglion cell loss after postgeniculate visual pathway injury.

Birth After Electroejaculation Coupled to Intracytoplasmic Sperm Injection in a Gun-Shot Spinal Cord-Injured Man

We describe the case of a man who, after a gun-shot wound, has become paraplegic. As a consequence of his spinal cord injury he developed infertility due to incomplete erection and anejaculation. After several unsuccessful penile vibratory stimulation attempts, it was possible to achieve ejaculation by means of transrectal electrostimulation. However, the total sperm motility count, the percentage of normal sperm morphology, and the vitality and hypo-osmotic swelling test scores were rather low, and in particular viability was strikingly decreased during sperm preparation. These parameters further decreased following electroejaculate, forcing us to use intracytoplasmic sperm injection instead of conventional in vitro fertilization for the treatment cycle. A normal pregnancy was achieved which resulted in birth of a healthy girl on 1st January 1997.

Ovarian hyperstimulation syndrome: a clinical report on 4894 consecutive ART treatment cycles.

BackgroundAlthough a large number of studies have been dedicated to ovarian hyperstimulation syndrome (OHSS) none gave full embryological and clinical outcomes comparing oocyte trigger with human chorionic gonadotrophin (HCG) versus with a gonadotrophin-releasing hormone (GnRH) agonist (Buserelin) in cases with suspicious OHSS. The aim of the present study was thus to analyze 4894 consecutive assisted reproductive treatment cycles to undercover associated risk factors for development of OHSS, and the effects of the use of Buserelin as ovulation trigger on embryological and clinical outcomes.MethodsIn the 51 cases that developed OHSS, ovulation trigger was performed with HCG as indicators were not suspicious for OHSS. These were compared against two types of groups: 71 cases where Buserelin was used for ovulation induction due to suspicious development of OHSS; and those remaining 4772 cases where ovulation trigger was currently performed with HCG (control).ResultsOf the cases treated with Buserelin the oocyte maturation rate and the ongoing pregnancy rate were significantly lower, with higher rates of ectopic pregnancy and newborn malformations, but none developed OHSS. Of the OHSS cases, 23 needed hospitalization, with no major complications.ConclusionsYoung age, lower time of infertility, lower basal follicle stimulating hormone levels, higher number of cases with female factor and polycystic ovarian syndrome, high number of follicles and higher estradiol concentrations were the risk factors found associated with OHSS. Cases with OHSS also presented higher follicle count but the estradiol levels were within the normal range. It thus remains to develop more strict criteria to avoid all cases with OHSS.

Embryological, clinical and ultrastructural study of human oocytes presenting indented zona pellucida

Human oocyte dysmorphisms attain a large proportion of retrieved oocytes from assisted reproductive technology (ART) treatment cycles. Extracytoplasmic defects involve abnormal morphology of the zona pellucida (ZP), perivitelline space and first polar body. The aim of the present study was to describe a novel dysmorphism affecting the ZP, indented ZP. We also evaluated the clinical, embryological and ultrastructural features of these cases. We evaluated all ART treatment cycles during 7 consecutive years and found 13 treatment cycles (six patients) with all oocytes presenting an indented ZP. In addition, these oocytes presented total or partial absence of the perivitelline space, absence of resistance to ZP and oolemma penetration during microinjection, and low ooplasm viscosity during aspiration. This novel described dysmorphism was recurrent and attained all oocytes in three cases that had more than one treatment cycle. When compared with controls, data showed significant low oocyte maturity (42% versus 81.6%) and high cycle cancellation (30.8% versus 8.5%) rates, normal degeneration (3.4% versus 6.3%) and fertilization rates (69% versus 69.5%), and low pregnancy (15.4% versus 33.3%) and live-birth delivery (7.7% versus 27.7%) rates per cycle. Ultrastructure analysis revealed a zona pellucida structure with large empty electrolucent regions, an outer ZP layer with an indented surface with protuberances and a thick inner ZP that obliterated the perivitelline space. There was evidence of exocytosis of ZP material by the oocyte. In conclusion, oocytes with this novel described dysmorphism (indented ZP) are associated with low maturity, pregnancy and live-birth delivery rates.