Tubal Fluid Research Articles

Endometriosis-induced alterations in mouse metaphase II oocyte microtubules and chromosomal alignment: a possible cause of infertility

To examine the effect of peritoneal fluid (PF) of patients with endometriosis on the cytoskeleton of metaphase II oocytes and correlate the results with the stage of endometriosis and the duration of infertility. Prospective-controlled study. Center for reproductive medicine at a tertiary-care hospital. Women with endometriosis (n=23) and tubal ligation/reversal (n=15). Peritoneal fluid obtained from 38 women (23 with endometriosis and 15 tubal ligation/reversal) after laparoscopy. Four hundred metaphase II oocytes were used: 165 frozen metaphase II oocytes were incubated in the PF of patients with endometriosis, 135 oocytes incubated in the PF of nonendometriosis patients (control subjects) and 100 oocytes incubated in human tubal fluid (HTF) media. Spindle abnormalities (microtubule and chromosomal) were evaluated by confocal imaging. In the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment (score ≥3), indicating severe damage compared with the control groups. The proportions of abnormalities in microtubule and chromosome alterations in endometriosis (67.9% and 63.6%, respectively) were significantly higher than for oocytes incubated with PF of the nonendometriosis group (24.4% and 14.8%) as well as the HTF group (13% and 13%). Oocyte cytoskeleton damage positively correlated with the duration of infertility and the stage of endometriosis. Alteration of oocyte cytoskeleton might be one of the causes of poor oocyte quality in patients with endometriosis.

In vitro maturation (IVM) of murine and human germinal vesicle (GV) stage oocytes using variations of a human fallopian tube epithelial feeder cell system

OBJECTIVE: To assess the maturation rate of murine and human GV oocytes using variations of a human tubal feeder cell system. DESIGN: Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or co-culture with primary (CP58) or immortalized (CP13IM) human tubal cells. Rates of maturation were compared between groups. MATERIALS AND METHODS: Tubal epithelial cells were isolated from salpingectomy specimens by digestion with DNase/Collagenase. One batch was immortalized (CP13IM). Mouse GV oocytes were isolated from 8 week-old mice 40 h after PMSG stimulation. Human GV oocytes (n=21) were obtained from the MGH IVF lab. Murine and human GV oocytes were cultured in HTF media alone or on feeder layers of CP58 and CP13IM cells. Maturation rates were assessed by standard morphological criteria and compared between groups. RESULTS: Maturation rates of GV oocytes under the various culture conditions are shown in table 1. Tabled 1Maturation stage (% of total) of GV oocytes with different culture conditionsMurine (n=210, 24 h)Human (n=21)Culture systemGVGVBDMIIMII (24 h)MII (48 h)HTF27.5021.2551.2512.537.5CP588.7513.7577.5062.575.0CP13 IM12.5017.5070.0060.060.0 Open table in a new tab For murine GV oocytes (n=210 in 3 independent experiments), the maturation rate to the MII stage was approximately 50% higher with CP58 cells and nearly 40% higher with CP13IM cells compared to media alone. For human GV oocytes, the rate of maturation to the MII stage was approximately 5-fold higher with both the CP58 and CP13IM feeder systems at 24 h and approximately 2-fold higher at 48 h. CONCLUSIONS: This study shows that co-culture with either primary or immortalized tubal cells improved IVM rates for murine and human GV oocytes, suggesting a promising new technique with future applications in clinical reproductive medicine or in basic science research, such as in the generation of new stem cell lines.

Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates

We conducted a retrospective review of all frozen ETs from May 2002 to October 2007 using our new vitrification technique in order to determine its efficacy. We found high pregnancy (70.5%; 79/112) and implantation (40.6%; 110/271) rates with the use of this new method.

Interactions between apoptotic signal transduction and capacitation in human spermatozoa

Capacitation of sperm is a prerequisite for successful fertilization, determined by hyperactivated motility, increased tyrosine phosphorylation (TyrP) and membrane changes. However, the exact molecular mechanism is not fully clarified. The calpain-calmodulin-system is essential for membrane fusion during capacitation. Recently, interactions with caspase (CP) activation, a main feature of apoptotic cells, were postulated. The objective of our study was to examine interactions between apoptosis signalling and the calpain-calmodulin-system during capacitation. Semen samples from 20 healthy donors were incubated in human tubal fluid at 37 degrees C, 5% CO(2) for 3 h without additives (control), with 3% BSA (capacitation), 10 microM calpain-inhibitor III, 20 microM CP-1 inhibitor or 20 microM calmodulin-antagonist. Capacitation was monitored by computer assisted sperm motion analyzer, chlortetracycline (CTC)-assay and western blot (TyrP). Activation of caspases and integrity of transmembrane mitochondrial potential (TMP) were evaluated by flow cytometry. Capacitation, as measured by CTC assay, increased TyrP levels and hyperactivation, resulted in inactivation of CP-9, CP-3 and improved integrity of the TMP. Inhibition of calpain and CP-1 during capacitation reduced the capacitation-related parameters, but did not lead to apoptosis. Inhibition of calmodulin resulted in blocking of capacitation and stimulation of apoptosis. Interaction of the capacitation and apoptosis signalling systems seems to enable the capacitation process by prevention of apoptosis.

The impact of peritoneal fluid from healthy women and from women with endometriosis on sperm DNA and its relationship to the sperm deformity index

To study the effect of peritoneal fluid (PF) from healthy women and from women with endometriosis on sperm DNA and its relationship to sperm morphology as assessed by the sperm deformity index (SDI). Experimental study. Research laboratory at an academic hospital. Healthy women undergoing laparoscopic tubal ligation and women with endometriosis. Aliquots of prepared sperm from 10 healthy donors were incubated with PF from healthy women undergoing laparoscopic tubal ligation (treatment 1, n = 10), with PF from patients with endometriosis (treatment 2, n = 10), and with human tubal fluid media with 10% bovine serum albumin (control, n = 10). Sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and SDI. There was a significant increase in sperm DNA damage at 24 hours compared with at 1.5 and 3 hours. The SDI scores significantly correlated with sperm DNA damage after 1.5 and 24 hours of incubation in all aliquots. Percentage normal morphology did not correlate with DNA damage. In vitro exposure of sperm to PF from patients with endometriosis is associated with significantly increased DNA damage. There is evidence of interdependence between the sperm morphology as assessed by SDI scores and DNA damage. The significant increase in sperm DNA damage observed after 24 hours of incubation may be clinically relevant.

Protective effect of fallopian tubal fluid against activated leucocyte-induced sperm DNA fragmentation: preliminary results

The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a viable pregnancy. Oxygen radicals (ROS) have been identified as one of the main factors responsible for the induction of sperm DNA damage. Spermatozoa are mainly protected against ROS-induced damage by seminal plasma. However, this protective effect disappears once spermatozoa enter the female genital tract. The fallopian tube mucosa may play a protective role against ROS-induced sperm damage. The main objective of this study was to determine whether human tubal explants and tubal fluid exert a protective effect on ROS-induced sperm DNA damage. Spermatozoa were exposed to tubal explants and/or tubal fluid in the presence of phorbol myristate acetate (PMA)-activated polymorphonuclear leucocytes or control medium and sperm DNA fragmentation was measured using the TdT-mediated dUTP-biotin nick end labelling (TUNEL) test. Exposure of human spermatozoa to PMA-activated leucocytes resulted in a 2-fold increase in sperm DNA fragmentation. Co-incubation of spermatozoa with tubal explants did not reduce this damage. However, pre-incubation of spermatozoa with tubal fluid resulted in a statistically significant reduction in sperm DNA fragmentation levels, comparable to those observed in control. In conclusion, tubal fluid appears to protect against activated leucocyte-induced sperm DNA fragmentation, thus preserving the integrity of the paternal genome.

Comparison of the effects on penetration capacity of human spermatozoa between using Ham's F-10 medium and a medium based on the composition of human tubal fluid*: Vergleich des Einflusses der Penetrationskapazität menschlicher Spermatozoen bei Verwendung

The penetration ability of human spermatozoa on zona-free hamster ovum when using Ham's F-10 (HF-10) medium was compared with that when using a medium based on the composition of human tubal fluid. Forty semen specimens were each divided into two aliquots; one aliquot was washed with and incubated in HF-10 medium and the other in HTF medium. The penetration rate of sperm with motility between 30 to 60 percent was significantly increased when HTF medium was used (67.9 +/- 6.9%) compared with the rate when HF-10 medium was used (49.2 +/- 7.3%) (P less than 0.05). This study shows that the ZFHO penetration ability of human spermatozoa with poor motility can be improved by using HTF medium as compared with the use of HF-10 medium.

PTP1B Dephosphorylates N-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

Vitrification of immature mouse oocyte using stepwise equilibration before or after in vitro maturation

Immature mouse oocytes before or after in vitro maturation (IVM) were vitrified by the use of either conventional one-step or three-step equilibration. Although vitrification after IVM using the stepwise method does not affect the cleavage rate, the early embryonic development was profoundly impaired in both protocols.

MR Imaging Findings of Hydrosalpinx: A Comprehensive Review

Hydrosalpinx is a common adnexal lesion that may occur either in isolation or as a component of a complex pathologic process (eg, pelvic inflammatory disease, endometriosis, fallopian tube tumor, or tubal pregnancy) that leads to distal tubal occlusion. On magnetic resonance (MR) images, hydrosalpinx appears as a fluid-filled C- or S-shaped tubular structure that arises from the upper lateral margin of the uterus. Although hydrosalpinx is most often seen on ultrasonographic images, it also may be delineated on multiplanar MR images. MR imaging also may be useful for determining the cause of a hydrosalpinx or its associated adnexal process by characterizing the nature of the contents of the dilated tube. Tubal fluid with high signal intensity on T1-weighted images is suggestive of hematosalpinx associated with endometriosis and tubal pregnancy. A thickened wall of a dilated fallopian tube that displays variable or heterogeneous signal intensity may be indicative of pyosalpinx as a component of a tubo-ovarian abscess. The presence of an enhancing solid mass within a dilated tube is suggestive of fallopian tube carcinoma, whereas enhancement of the dilated tubal wall surrounding a saclike cystic mass may be indicative of a tubal pregnancy. Understanding the pathogenesis and clinical manifestations of conditions associated with hydrosalpinx may aid in the timely diagnosis of complex adnexal masses at MR imaging, enabling avoidance of unnecessary procedures.

Comparison of survival rate of cleavage stage embryos produced from in vitro maturation cycles after slow freezing and after vitrification

Significantly more embryos survived the vitrification procedure compared to slow freezing (85.5% vs. 61.8%) in cleavage-stage human embryos produced from in vitro maturation cycles, suggesting that vitrification is more efficient than slow freezing for cryopreservation.

271 THE EFFECT OF TEMPERATURE ON HUMAN TESTICULAR TISSUE TO OPTIMIZE THE IN VITRO MATURATION OF PRE-FREEZE MOTILITY

The development of intracytoplasmic sperm injection has made the use of testicular sperm a viable option for infertile men with obstructive and nonobstructive azoospermia. Over the past decade, testicular biopsies have been handled and processed using a variety of different methods. Whole biopsy pieces can be effectively cryopreserved in a 10% glycerol diluent (Schiewe et al. 1997 67, S115 abst); however, the ability to find viable, motile sperm post-thaw is improved when prefreeze motility exists. The purpose of this study was to comparatively document in vitro sperm motility enhancement over time at different temperatures, and to prove that an intermediate temperature (28 to 30°C) would optimize sperm longevity for up to 1 week. In this study, 10 men with obstructive azoospermia underwent a surgical, open testicular biopsy procedure. Each biopsy was placed in HEPES buffered-human tubal fluid (mHTF) medium supplemented with 5% human serum albumin (HSA; Irvine Sci., Santa Ana, CA, USA) and transported to the laboratory at room temperature. Each testis biopsy (TBx) was dissected into 8 equal pieces (approximately 2 × 2 × 1 mm). Five intact pieces of TBx were cryopreserved in separate cryovials for future use. The remaining TBx tissue was subdivided into 1 of 3 temperature treatment groups (24, 30, or 37°C) for extended IVM. The 30°C incubation condition was achieved by placing the dish(es) in a Styrofoam box placed on a 37°C warming plate. Each TBx was placed into a separate 100 × 35 mm Falcon dish in 150-μL droplets of mHTF under oil and shredded by needle dissection to disperse the contents of the seminiferous tubules. Reminant tissue was placed into another droplet for additional dissection, as needed. Sperm were analyzed for motility (graded as Type I = twitching, II = undulating, III = slow progression, and IV = rapid progression) at 0, 24, 96, and 144 h without replacing the IVM medium. The percentage increase in motility, compared with 0 h, was statistically contrasted across temperature treatments by chi-squared analysis. Testicular sperm motility ranged from 5 to 25% at 0 h and increased in all groups at 24 h. There was no difference in total motility at 24 h, but progressive motility was higher (P < 0.05) at 37°C compared with 24°C. Significant differences (P < 0.05) were observed in total percentage of motility/percentage of progressive motility at 96 h with treatment differences (*) being 30°C (66%*/44%*) > 24°C (42%*/14%) > 37°C (18%*/10%). This statistical trend continued at 144 h with 30°C (42%*/23%*) > 24°C (24%*/8%) > 37°C (9%*/5%). During this study, a viable pregnancy was achieved using a 30°C sample 8 days post biopsy, exceeding 2 previous healthy triplet pregnancies, which were successful using 4-day-old TBx specimens in 1996 and 2004. This study confirms that an intermediate IVM temperature of 30°C is optimal to enhance TBx cryopreservation, which in our laboratory is generally performed at 24 to 72 h of IVM. Our routine pregnancy success in applying ICSI with IVM, thawed TBx sperm discounts the concern some individuals express regarding DNA fragmentation of sperm over time.

주화성에 의한 활동성 정자 분리용 마이크로 바이오칩에 관한 연구

This paper presents a new microchip which can separate motile sperm by chemotaxis. The microchip was developed to create longitudinal concentration gradient in the microchannel due to diffusion. Linearly good concentration gradient of chemoattractant was generated without any fluid control devices. In sperm separation experiment with the developed microchip, mouse sperm was used as sample and acetylcholine was selected as chemoattractant. Human tubal fluid (HTF), buffer solution, was introduced into the microchannel of the microchip and attractants diluted in ratio of 1, 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 including control (DI water) were dropped in each outlet by 2 ㎕ volume with micropippet. After 5min, 1 ㎕ sperm solution was dropped into inlet of the chip. After 10 min, when sperms reached to the outlet by chemotaxis, we counted sperms in each outlet by using microscopy. Consequently, we could separate progressive motile sperm with the new microchip. In the experiment, the most sperms were isolated at the outlet dropped with 1/16 diluted solution. The optimal concentration gradient to induce chemotaxis was about 0.625 ㎎/㎖/㎜.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Vaginal administration of mifepristone: effectiveness and benefits

OBJECTIVE: To investigate the cytotoxicity and contraceptive effects of mifepristone on animals receiving the drug via different administration routes.DESIGN: Administrate mifepristone through vaginal and oral to animal, collect respective related experiment result and compare their contraception rate and cytotoxicitity.MATERIALS AND METHODS: (1) Mifepristone was delivered to rabbits (6 mg/kg) by the vaginal or the oral administration. Serum concentrations of the drug, and two female hormones estradiol and progesterone were measured at different times. Ca2+ concentrations in the endocrine tubal fluid and tissues were also examined. (2) Effects of mifepristone delivered by the two different routes on the contraception of the animal were compared. (3) Cytotoxic effects of mifepristone on rat livers and kidneys were also investigated by light microscopy.RESULTS: (1) Comparing animals administrated the drug orally with those by the vaginal route, serum concentrations of mifepristone and estrogen were both significantly lower, but Ca2+ contents and the frequencies of smooth muscle contraction in the tubal tissues had no significant difference. (2) Both vaginal and oral administrations of the drug achieved good contraception, with the success rates of 80% and 83%, respectively. (3) Mifepristone caused certain liver and kidney damage in animals receiving the drug orally, but did not induce any significant pathological changes in animals receiving the drug via vagina.CONCLUSIONS: Mifepristone administrated via vagina resulted in the higher plasma concentrations of the drug, thus potentially can be used at the reduced doses. While both vaginal and oral administrations were able to achieve good contraceptive effects, the vaginal route showed no cytotoxicity to the liver and kidney. OBJECTIVE: To investigate the cytotoxicity and contraceptive effects of mifepristone on animals receiving the drug via different administration routes. DESIGN: Administrate mifepristone through vaginal and oral to animal, collect respective related experiment result and compare their contraception rate and cytotoxicitity. MATERIALS AND METHODS: (1) Mifepristone was delivered to rabbits (6 mg/kg) by the vaginal or the oral administration. Serum concentrations of the drug, and two female hormones estradiol and progesterone were measured at different times. Ca2+ concentrations in the endocrine tubal fluid and tissues were also examined. (2) Effects of mifepristone delivered by the two different routes on the contraception of the animal were compared. (3) Cytotoxic effects of mifepristone on rat livers and kidneys were also investigated by light microscopy. RESULTS: (1) Comparing animals administrated the drug orally with those by the vaginal route, serum concentrations of mifepristone and estrogen were both significantly lower, but Ca2+ contents and the frequencies of smooth muscle contraction in the tubal tissues had no significant difference. (2) Both vaginal and oral administrations of the drug achieved good contraception, with the success rates of 80% and 83%, respectively. (3) Mifepristone caused certain liver and kidney damage in animals receiving the drug orally, but did not induce any significant pathological changes in animals receiving the drug via vagina. CONCLUSIONS: Mifepristone administrated via vagina resulted in the higher plasma concentrations of the drug, thus potentially can be used at the reduced doses. While both vaginal and oral administrations were able to achieve good contraceptive effects, the vaginal route showed no cytotoxicity to the liver and kidney.

Are testicular sperm in epididymal necrospermia (EN) normal?

OBJECTIVE: Evaluate the viability of testicular sperm in patients with idiopathic EN. DESIGN: Prospective clinical analysis. MATERIALS AND METHODS: Semen and testicular samples from three men with consistent absolute asthenospermia (0% motility) and between 70 to 100% necrospermia in the ejaculate were studied under electron microscopy (EM) after ruling out bacterial infections. The female partners of all three patients underwent controlled ovarian hyperstimulation (COH) for intracytoplasmic sperm injection (ICSI) with testicular sperm extraction (TESE) that was undertaken at the time of follicular aspiration. Scrotal exploration was performed under local anesthesia through a small incision and the samples obtained were were placed in modified human tubal fluid (HTF) culture media. The seminiferous tubules were dispersed with the aid of fine needles under an inverted microscope. All semen and testicular samples were studied under phase-contrast microscopy at X400. After dilution and centrifugation the pellets were fixed with 3% glutaraldehyde, post fixed in 1.3% osmium tetroxide and embedded in Epon-Araldite for electron microscopy scanning. RESULTS: Motile spermatozoa were harvested from TESE samples in all patients and five ICSI cycles were performed. Fertilization rate was 64% (9/14) (Patient 1), 83% (5/6) (Patient 2) and 79% (15/19) (Patient 3) in MII oocytes. We obtained two pregnancies in the same patient (Patient 3): an early abortion in the first attempt and a term twin pregnancy after transfer of three cryopreserved embryos. EM studies of ejaculated spermatozoa showed no evidence of flagellar abnormalities and autolysis of all tails and great part of the spermatic heads in two patients; and axonemal absence in all tails studied in one patient, suggesting a post-necrotic phenomenon. EM studies of testicular sperm showed normal flagellar configuration and the presence of axonemes without degenerative signs. CONCLUSIONS: It has been proposed that in EN spermatozoa are produced normally in the testis and that death of the sperm cells occurs during transit through the epididymus, either during storage or when contacting seminal fluid. Our patients had testicular spermatozoa with normal fertilization potential. The EM findings coupled with the fact that a normal twin birth was achieved through cryopreserved embryo transfer, suggests that testicular spermatozoa from patients with severe necrospermia in the ejaculate are, indeed, normal.

Nitric oxide, antioxidants, and mitochondria activity for preimplantation development

OBJECTIVE: Nitric oxide (NO) is an important regulator of apoptosis for early embryo development and a documented modulator of mitochondrial activity for several types of somatic cells. This study attempts to elucidate the effect of NO on mitochondrial activity for early cleavage stage embryos. Furthermore, the capability of antioxidants to against the apoptotic effect of NO for preimplantation embryos is also investigated. DESIGN: Prospective, controlled study. MATERIALS AND METHODS: Zygotes from superovulated B6CBF1 female mice were retrieved from excised oviducts and cultured within modified human tubal fluid (mHTF) medium. Sodium nitroprusside (SNP) and NG-nitro-L-arginine (L-NA) were supplemented into the culture medium and functioned as NO donor and NOS inhibitor, respectively. Mitochondrial membrane potential and amount of ATP were measured for monitoring mitochondrial activity. Mn(III) tetrakis(4-benzoic acid) porphyrin choloride (TBAP), ebselen (EBS), and glutathione methyl ester (GSH) were applied into the culture medium to reduce oxidative stress. Apoptosis assay for blastomeres was performed with terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay. The blastocyst formation rate and number of apoptotic cells were recorded as an index of embryo development. RESULTS: Supplementation of SNP impaired embryo development and induced disruption of mitochondrial membrane potential in a dose-dependent manner. Although L-NA also arrested embryo development, L-NA did not break the integrity of mitochondrial membrane potential. The degenerated embryos after NO exposure exhibited apoptosis demonstrated by TUNEL stain. Subsequent to NO exposure, the addition of TBAP (superoxide scavenger) or GSH was able to rescue the embryos. However, the supplementation of TBAP was associated with a decline of blastocyst formation rate. Furthermore, EBS (peroxynitrite scavenger) was not able to rescue the embryos from NO toxicity. CONCLUSIONS: The NO donor exerted deleterious effect on embryo development, which might through disruption of mitochondrial membrane potential and induced mitochondria-dependent apoptosis. In order to counteract this adverse effect, GSH might be a required addictive of culture medium.

Relationship between extent of cytoskeleton damage of oocytes incubated in PF of endometriosis and the stage of the disease and duration of infertility

OBJECTIVE: Our previous studies demonstrated that endometriosis may induce alterations in the cytoskeleton (microtubules and chromosomes). Our objective was to evaluate the relation between the degrees of damage of the cytoskeleton of oocytes incubated in PF of patients with endometriosis and the stage of the disease as well as the duration of infertility. DESIGN: Experimental prospective controlled study. MATERIALS AND METHODS: PF was collected from patients with endometriosis and divided into: Group I = mild (stage 1 and 2) and group II = severe (stages 3 and 4). 400 mature metaphase II mouse oocytes were divided into three groups. Group I: 165 incubated in the PF of patients with endometriosis both mild and severe disease. Group II: 135 incubated in the PF of patients with tubal ligation (controls) Group III: 100 Oocytes incubated in plain human tubal fluid media (HTF). Oocytes were fixed in Formaldehyde 3 % followed by incubation with anti α-tubulin monoclonal antibody for microtubule staining and FITC labeled anti-mouse Ig G antibody. Propidium iodide was used for chromosomal staining. Microtubules and chromosomes were examined by confocal microscopy. Both microtubules and chromosomes were scored as: 1-2 = normal and 3-4 = abnormal. RESULTS: Oocytes that were incubated in PF of patients with endometriosis showed high grade of microtubules and chromosome damage compared to control. Percentage of abnormal microtubules/chromossome score were seen in endometriosis (68/64) vs. tubal ligation PF (24/19) and HTF) (18/18) (p< 0.001 for all groups compared with control. The degree of damage for microtubules correlated with stage of the disease (r = 0.42; P<0.029) and duration of infertility (r = 0.46, p<0.001) and for chromosomes scores, for stage (r = 0.17; p<0.029) and duration (r = 0.20; P< 0.014, respectively).Table 1Microtubules and chromosomes damage in patients and controlScoresControl (Tubal ligation)Control (HTF)EndometriosisP-valueMicrotubules (mean ± SD) % abnormal2.07 ± 0.85 24%1.95 ± 0.72 18%2.81 ± 0.86 68%<0.001Chromosomes (mean ± SD) % abnormal1.89 ± 0.82 19%1.84 ± 0.75 18%2.72 ± 0.72 64%<0.001P<0.05 was significant between control and endometriosis group. Open table in a new tab P<0.05 was significant between control and endometriosis group. CONCLUSIONS: Endometriosis damages microtubules as well as the chromosomes of metaphase II mouse oocytes, which may lead to poor oocyte quality. The degree of damage is correlated to the stage of the disease and the duration of infertility.

In vitro fertilization in inbred BALB/c mice II: effects of lactate, osmolarity and calcium on in vitro capacitation

To elucidate requirements for in vitro sperm capacitation in inbred BALB/c mice, osmolarity, calcium and lactate were optimized using modified simplex optimization medium (mKSOM). Modified human tubal fluid (mHTF), a capacitation-supporting medium, was used as a control. In the first series of experiments, the effects of calcium and osmolarity were studied in the presence of lactate. Although preincubation with >or=5 mM CaCl2 improved fertilization after insemination significantly, it was still significantly lower than incubation with mHTF. To obtain fertilization at the equivalent levels to that of mHTF, isotonic osmolarity (305 mOsmol) was required. Trehalose, an osmotic reagent, could substitute for NaCl partially. In the second series of experiments, the effects of lactate were examined using a concentration of 5 mM calcium and isotonic osmolarity. Preincubation with <or=2.5 mM lactate increased fertilization significantly (>75%), as well as the percentages of B (capacitated) pattern sperm (>or=40%) in chlortetracycline (CTC) staining, as compared with incubation in mHTF (46% and 28%, respectively; p<0.05). In the third series of experiments, the effects of osmolarity and calcium in the absence of lactate were examined. An increase in osmolarity during sperm preincubation increased both fertilization and B-pattern sperm significantly in a dose-dependent manner. Trehalose, sucrose and choline chloride could substitute for NaCl. An increase in CaCl2 concentration during preincubation had no effect on fertilization, but this increase reduced the percentages of B-pattern sperm. In vitro capacitation of inbred BALB/c mice is sensitive to lactate and osmolarity, but that sensitivity for calcium varies depending on the presence or absence of lactate.

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

To optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm-ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230-305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25-75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.