Semen Analysis of Total Motile Sperm Count Based on the 1999 and 2010 WHO Criteria.

OBJECTIVE: Approximately 15% of the couples suffer from infertility. Half of the cases of infertility are due to male factors. Several sperm function tests have been proposed to evaluate male fertility, but sperm analysis is still the first and most important diagnostic test for male infertility. The prognostic value of semen characteristics such as concentration, morphology and motility markers are often confused with male infertility. Evaluation of seminal parameters and classification for normality remains a frequent topic of discussion. METHODS: This study evaluated 477 semen samples from men undergoing investigation or infertility treatment between 2011 and 2015. RESULTS: The spermograms of 401 patients were deemed abnormal based on the 1999 World Health Organization (WHO) criteria; the number changed to 223 when the spermograms were assessed based on the 2010 WHO criteria and to 200 when Total Motile Sperm Count (TMSC) was used as the criterion. Sperm morphology was the item in the criteria that most significantly changed spermogram classification. Normality parameters became less rigid from 1999 to 2010, thereby significantly changing the proportion of individuals no longer described as infertile/subfertile. CONCLUSIONS: The classification based on TMSC could not differentiate between fertile and infertile subjects for not taking sperm morphology into account. Nevertheless, it may be helpful in cases where intrauterine insemination is indicated.

Ceruloplasmin, serotransferrin and albumin presented different abundance in mares' uterine fluid five days after insemination.

Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-dimensional electrophoresis (2D-PAGE). A total of 107 spots were visualized by 2D-PAGE. Three spots with differences in abundance between the inseminated and cyclic mares and with presence in at least 80% in one of the groups were selected and identified. The selected spots were extracted, digested by trypsin and analyzed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) for protein identification. Three proteins were identified: ceruloplasmin (CP) serotransferrin (TF) and albumin (ALB). The identified proteins in this study were related to iron metabolism and immunological tolerance suggesting that changes in their abundance during the 5 days after ovulation are probably a signaling mechanism of the potential equine conceptus to the maternal immune system for its immunological recognition. Probably changes in abundance of CP, ALB and TF represent a mechanism of endometrial preparation for the maternal recognition, attachment and development of a potential equine embryo. There is also evidence to support an alternative hypothesis suggesting that protein changes are inflammatory events, resulting from a previous inflammation due to residual seminal effects.

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus-oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 µM BCB for 60 min; and 60, 90 or 120 min to 13 µM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 µM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 µM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 µM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

Developmental potential of in vitro or in vivo matured oocytes.

This study compared the embryological features of mature and immature oocytes (different stages) collected from stimulated cycles of in vitro fertilization (IVF). Immature oocytes were identified, classified as PI (prophase I - germinal vesicle, GV) or MI (metaphase I), were matured in vitro and fertilized using the intra-cytoplasmic sperm injection (ICSI) technique. Fertilization potential, cleavage, and subsequent transfer/cryopreservation of the embryos derived from these in vitro matured oocytes were compared with those of in vivo matured oocytes (collected at the MII stage). The characteristics of embryos derived from gametes recovered in the MI and MII stages were similar. The fertilization rate of immature oocytes recovered in PI was significantly lower than that of MII oocytes (P = 0.031), and the cleavage rate of the PI group was also lower than that of the MI (P = 0.004) and MII (P < 0.001) groups. In vitro maturation of MI oocytes is a suitable alternative when immature oocytes are recovered, as their characteristics and development are similar to those of in vivo matured oocytes. Optimization of outcomes for PI oocytes will require development of techniques that can distinguish which of these gametes will mature and fertilize.

Human granulosa cells: insulin and insulin-like growth factor-1 receptors and aromatase expression modulation by metformin.

BACKGROUND/AIM: Granulosa cells are the source of the most important ovarian steroids. Even in patients without significant improvement in metabolic parameters, metformin has apparently an important effect on the ovary. The aim of this study was to evaluate gene and protein expression of an insulin receptor (IR), insulin-like growth factor-1 (IGF1) receptor (IGF1R) and aromatase in granulosa cells treated with metformin and insulin. METHODS: Luteinized granulosa cells were collected from 27 patients during in vitro fertilization procedures. Cells were isolated, stored in culture for 24 h and divided into four groups: control; metformin for 30 min, and metformin for 30 min plus insulin for 30 or 60 min. RESULTS: IR and IGF1R mRNA expression was significantly enhanced by metformin but was not affected by insulin. Aromatase mRNA expression was significantly reduced in metformin-incubated cells following stimulation with insulin for 30 min. No statistical differences were found in IGF1R and aromatase protein expression, and IR expression was not detected. CONCLUSION: A direct effect of metformin on the gene expression of IGF1R, IR and aromatase was observed. Further studies should investigate the role of IGF1R, IR and aromatase in ovarian physiology for a better understanding of the effect of metformin.

Mechanical processing of hyperviscous semen specimens can negatively affect sperm DNA fragmentation.

INTRODUCTION: The present study compared the DNA fragmentation in human sperm samples with reduced, physiological, and increased viscosity in order to evaluate whether the process used to reduce viscosity (expulsion of semen through a needle and syringe) alters significantly sperm DNA fragmentation. METHODS: The seminal parameters of semen samples from 123 patients were evaluated and classified according to their viscosity. Samples with increased viscosity were submitted to a process of expulsion of semen through a 10-mL syringe and an 18-gauge (18G) needle to reduce the seminal viscosity. The DNA fragmentation of all samples was analysed using TUNEL assay (Terminal deoxynucleotidyl transferase mediated dUTP Nick-end labelling assay); in samples with increased viscosity, the fragmentation was assessed before and after the process of expulsion with syringe and needle. RESULTS: There was no difference in DNA fragmentation between groups with different viscosity (P = 0.857). A significantly increase in sperm DNA fragmentation after expulsion of hyperviscous semen through the syringe was observed (P = 0.035). CONCLUSION: There was no difference in DNA fragmentation rate between samples with reduced, increased and physiological viscosities; however, the physical process of expulsion of semen through a syringe and needle increased sperm DNA fragmentation.

Impact of tubal ligation on ovarian reserve as measured by anti-Müllerian hormone levels: a prospective cohort study.

BACKGROUND: Tubal ligation (TL) is considered a very efficient contraceptive method. However, some patients complain of a variety of postoperative symptoms. The objective of this study was to investigate whether ovarian reserve may change after tubal ligation. STUDY DESIGN: This was a prospective cohort study of 80 fertile women who underwent TL. Ovarian reserve was evaluated by measuring pre- and postoperative (1 year after surgery) serum anti-Müllerian hormone (AMH) levels and transvaginal antral follicular count (AFC). Potential confounding factors were age, body mass index (BMI), smoking, surgical technique and prior contraceptive methods. The Wilcoxon test was used to compare pre- and postoperative (12 months) AMH and AFC, and simple and multiple linear regression were used to evaluate confounding factors. RESULTS: Fifty-two patients completed the study protocol. The median AMH level was 1.43 ng/mL (interquartile range 0.63-2.62) preoperatively and 1.30 ng/mL (interquartile range 0.53-2.85) after 12 months (p=.23). The mean AFC was 8.0 (interquartile range 5.0-14.0) and 11.0 (interquartile range 7.0-15.0) before and after TL, respectively (p=.12). These differences were not statistically significant. Use of a hormonal contraceptive method prior to TL was significantly associated with a postoperative increase in AMH. CONCLUSIONS: This study suggests that ovarian reserve is not altered by TL.

Adequacy of ovarian diathermy under ultrasound control: an experimental model.

BACKGROUND: To develop a minimally invasive ovarian cauterization technique under transvaginal ultrasound control and evaluate the safety and feasability of monopolar cauterization to cause ovarian injury using female cattle of reproductive age as an experimental model. METHOD: Experimental study in a university research center was performed. Eleven female bovines of reproductive age were submitted to monopolar transvaginal ovarian cauterization. The right ovary (RO) was punctured at four sites and 40 W was applied for 5 s at each point, resulting in a total of 800 J (Joules) of thermal energy. In the left ovary (LO), the procedure was similar, with the same time and 80 W, resulting in a thermal energy of 1600 J. Macroscopic and microscopic lesions were assessed. RESULTS: Of 22 ovaries punctured, 20 were cauterized and exhibited macroscopic and typical microscopic lesions. No lesions could be found in the needle path. The measures of the areas of microscopic electrocautery lesions calculated estimating a cylindrical volume showed a median of 1.12% in the right ovary and 1.65% in the left ovary. When the estimate was calculated by spherical shape, the medians were 1.77% in the right ovary and 3.06% in the left ovary. There was a statistically significant difference in these two estimates (sphere, p = 0.008; cylinder, p = 0.021). CONCLUSION: The experimental animal model described for transvaginal ultrasound-guided ovarian needle cauterization seems to be feasible. The ovaries were successfully cauterized without injuries in needle path and more energy resulted in significantly more thermal lesion. The safety and effectiveness of this technique, theoretically less invasive than current ovarian drilling methods, could be tested in anovulatory women with PCOS.

Transvaginal ultrasound ovarian diathermy: sheep as an experimental model.

BACKGROUND: Some techniques of transvaginal ovarian drilling have been previously described. Nevertheless a monopolar transvaginal ovarian cauterization, that use the expertise and safety of transvaginal puncture for oocyte captation seems to be an easier and feasible approach. The aim of this study was to develop a minimally invasive ovarian cauterization technique under transvaginal ultrasound control, and to evaluate the safety of the transvaginal ovarian monopolar cauterization, female sheep at reproductive age were used as an experimental model. FINDINGS: An experimental study was performed in a university research center. Seventeen female sheep (15 Corriedale e 2 Suffolk) in reproductive age were submitted to transvaginal ovarian cauterization with a monopolar Valleylab Force 2 electrocautery. Macroscopic and microscopic lesions were assessed. Ovarian size were 1.31 cm2 ± 0,43 (Corriedale) and 3.41 cm2 ± 0,64 (Suffolk). From 30 ovaries from Corriedale sheep punctured, only 3 were cauterized, presenting macroscopic and typical microscopic lesion. In the Suffolk sheep group, only one ovary was cauterized. No lesion could be found in the needle path. CONCLUSIONS: This is the first experimental animal model described for ovarian cauterization needle guided by transvaginal ultrasound. The sheep does not seem to be the ideal animal model to study this technique. Another animal model, whose ovaries are better identified by transvaginal ultrasound should be sought for this technique, theoretically less invasive, before it could be offered safely to women with polycystic ovary syndrome.

Proporção macho: fêmea de embriões bovinos cultivados na presença ou ausência de glicose após FIV com espermatozóides selecionados por Swim-up ou Gradiente de Percoll

No sistema de PIV em bovinos, tem sido obtida uma elevada porcentagem de embriões machos. Este experimento foi realizado para determinar se a presença de glicose no meio de cultivo afeta a proporção macho:fêmea (M:F) dos embriões bovinos PIV a partir da FIV com espermatozóides preparados pelos métodos do swim-up (S) ou do gradiente de Percoll (P). Após a MIV, os COCs foram divididos em dois grupos e inseminados com espermatozóides preparados por um dos métodos. Os zigotos foram cultivados em meio com ou sem 5,56mM de glicose, totalizando 4 tratamentos: S-Gli, S+Gli, P-Gli e P+Gli e 48h após a inseminação, os embriões de cada tratamento foram submetidos à sexagem por PCR (n=845). O efeito da glicose no meio de cultivo sobre a proporção M:F dos embriões PIV a partir dos dois métodos foi semelhante (teste do c²), resultando em uma porcentagem de machos menor do que 50 por cento no estágio de 2-C (S: 30,8 por cento; P: 23,8 por cento: P<0,01) e maior do que 50 por cento no estágio de 8-C (S: 79,4 por cento; P: 68,8 por cento: P<0,01). Estas porcentagens foram diferentes (P<0,05) das observadas quando os embriões foram cultivados sem glicose, tanto no estágio de 2-C (S: 48,5 por cento; P: 41,5 por cento) como no de 8-C (S: 62,5 por cento; P: 50,8 por cento). A presença de glicose não afetou a proporção M:F no total de embriões produzidos (S: 56,7 por cento; P: 49,0 por cento), que foi semelhante à observada na ausência de glicose (S: 55,7 por cento; P: 46,2 por cento). Portanto, a glicose exacerbou a diferença na velocidade de desenvolvimento entre os embriões machos e fêmeas.