Peritoneal fluid prostanoids in unexplained infertility

Elevated peritoneal fluid thromboxane B2 and 6-keto-prostaglandin F1 alpha have been reported in patients with biopsy-proved endometriosis. In this paper these peritoneal fluid prostanoids were measured in patients with unexplained infertility and a fertile control group matched for age and day of the menstrual cycle. A subgroup of the women with unexplained infertility demonstrated a marked elevation of both prostanoids (p less than 0.01). These elevations may serve to identify women in whom undetected endometriosis or some other peritoneal irritant is associated with infertility.     

Effect of peritoneal fluid on in vitro cleavage of 2-cell mouse embryos: possible role in infertility associated with endometriosis

The purpose of this study was to evaluate the effect of peritoneal fluid (PF) on in vitro cleavage of 2-cell mouse embryos. PF was aspirated from the posterior cul-de-sac at laparoscopy and centrifuged, and the cell-free supernatant was heat-inactivated at 56 degrees C for 30 minutes and filtered (0.22 micron). Five percent PF in Ham's F-10 media was prepared and eight to ten 2-cell mouse embryos cultured for 72 hours. There were two study groups, one consisting of 10 PF samples from infertile patients with no endometriosis (PF-NE) and 18 from infertile patients with endometriosis (PF-E). Each sample was assayed along with a control consisting of media only. At 72 hours, greater than 50% of the embryos in the control groups reached the blastocyst and hatching stages. Individual PF samples in both study groups were classified as toxic if less than 50% of the embryos reached the blastocyst and hatching stages at 72 hours. Eight of the 10 samples in the PF-NE group were nontoxic and 14 of the 18 samples in the PF-E group were toxic (P less than 0.01). For evaluation of the overall effect of PF, all results in the PF-E group were pooled (162 embryos) and compared by the Mann-Whitney U test to pooled results in the PF-NE group (100 embryos). The embryonic stages in the PF-NE group were significantly more advanced than those in the PF-E group (P less than 0.001) at 24, 48, and 72 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peritoneal fluid prostaglandins in endometriosis, tubal disorders, and unexplained infertility

To elucidate the roles of prostaglandins in peritoneal fluid and sex steroids in patients with endometriosis (N = 29), tubal disorders (N = 15), and unexplained infertility (N = 13), assays were performed using 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (a metabolite of prostacyclin), thromboxane B2 (a metabolite of thromboxane A2), estradiol, and progesterone. Women with normal pelvic anatomy (N = 25) served as controls. Peritoneal fluid 6-keto-PGF1 alpha concentrations in patients with endometriosis (742 +/- 104 pg/ml, mean +/- SE), tubal disorders (987 +/- 211 pg/ml), and unexplained infertility (1659 +/- 770 pg/ml) were higher than those in the control women (515 +/- 77 pg/ml). The thromboxane B2 levels in the peritoneal fluid in endometriosis (554 +/- 73 pg/ml), tubal disorders (614 +/- 107 pg/ml), and unexplained infertility (668 +/- 161 pg/ml) were higher than the levels in the control subjects (333 +/- 23 pg/ml). There was no relationship between 6-keto-PGF1 alpha/thromboxane B2 in peritoneal fluid and day of menstrual cycle. The concentrations of estradiol and progesterone were normal in all patient groups and were not related to the 6-keto-PGF1 alpha and thromboxane B2 levels. The authors suggest that these prostanoids, which may contribute to infertility, may originate mainly from the peritoneum as a result of irritation by endometriotic implants, tubal adhesions, and scarring.     

Peritoneal fluid fractions from patients with endometriosis do not promote two-cell mouse embryo growth

Peritoneal fluid (PF) from 10 infertile patients with endometriosis, obtained during the follicular phase of the cycle during laparoscopy, did not promote two-cell mouse embryo growth to the extent observed by fluid obtained from seven normal controls. Five molecular weight (MW) fractions were obtained by ultrafiltration, and each was used as media supplement in the assay and compared with PF fractions from normal controls. All fractions of PF from patients with endometriosis inhibited mouse embryo growth to a greater extent than did normal controls. However, the MW fractions greater than 100,000 daltons showed greater inhibition of embryo development than did fractions less than 100,000 daltons. This study of cell-free PF suggests the presence of a humoral factor greater than 100,000 daltons that is inhibitory on mouse embryo growth.     

Peritoneal fluid and serum steroids in infertility patients

Peritoneal fluid and serum were collected from 78 patients at the time of laparoscopy. Twenty-two were fertile controls (CTL), and 56 were infertility patients, who were subdivided into three main groups: endometriosis (EMS), pelvic adhesions (ADH), and ovarian dysfunction (OvDF). Based on control group data, biochemical criteria indicative of the presence of a stigma, S(+), were established: (1) serum progesterone (P) greater than or equal to 2 ng/ml, (2) peritoneal fluid P greater than or equal to 50 ng/ml, and (3) peritoneal fluid/serum ratio of P greater than or equal to 3. Direct visualization by laparoscopy showed that 21% CTL, 75% EMS, 69% ADH, and 56% OvDF subjects had luteinized unruptured follicle (LUF) syndrome. Biochemical criteria, however, demonstrated only 7% CTL, 37% EMS, 23% ADH, and 56% OvDF subjects had LUF. Peritoneal fluid estradiol (E2) and P concentrations and total content were significantly lower in LUF than in non-LUF patients, whereas serum E2 and P concentrations were not different between the two groups. Values for testosterone and androstenedione in peritoneal fluid and serum were similar between these two groups. Endometrial dating in LUF versus non-LUF patients were also similar. The usual indicators of ovulation, i.e., serum P, endometrial dating, and basal body temperature, failed to identify LUF. The diagnosis of LUF can be best made by P assay of peritoneal fluid and serum.     

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

Purpose

The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.

Methods

Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.

Results

Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P?>?0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P?<?0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P?<?0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P?<?0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P?>?0.05).

Conclusions

Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.     

The concentration of interleukin-2 and interleukin-2 soluble receptors in peritoneal fluid of patients with unexplained infertility

OBJECTIVES: We measured the concentration of interleukin-2 (IL-2) and interleukin-2 soluble receptor (sIL-2R) in peritoneal fluid (PF) of patients with unexplained infertility. MATERIALS AND METHODS: PF was obtained during laparoscopy from 7 women with unexplained infertility (UI) and 11 women with benign noninflammatory ovarian tumors. All laparoscopies were performed in follicular phase of the cycle. IL-2 and sIL-2R concentrations were measured in PF supernate stored in -70 degrees C until analysis using ELISA method (ENDOGEN). RESULTS: We found significantly (p = 0.009) lower concentration of sIL-2R in PF from patients with UI (303.844 U/ml) than in reference group (556.385 U/ml). The level of IL-2 was not detectable in 2 cases from women with UI and 5 cases from reference group. The concentration of IL-2 in PF did not differ (p = 0.135) between patients with UI (2.346 pg/ml) and those from reference group (1.064 pg/ml). CONCLUSIONS: The concentration of sIL-2R in PF of patients with UI was lower than in those from reference group. This may be the factor responsible for insufficient local immunosuppression, affecting reproduction.     

Rescue of mouse embryos from 2-cell blocks by microinjection of maturation promoting factor

Objective: To examine the rescue of mouse embryos from 2-cell blocks by the microinjection of maturation promoting factor (MPF) extracted from matured Xenopus eggs into one of the blastomeres of 2-cell stage mouse embryos.

Design: Controlled laboratory study.

Setting: First Department of Obstetrics and Gynecology, Toho University School of Medicine, Tokyo, Japan.

Animal(s): Eight- to 10-week-old female Crj:CD-1(ICR) mice.

Intervention(s): One of the blastomeres of the mouse 2-cell embryos was injected with MPF (MI group) or mHTF medium (MED group) at 28–32 hours after insemination.

Main Outcome Measure(s): The developmental rate to blastocyst.

Result(s): The developmental rate to blastocyst in the MI group (48.0%) was significantly higher than that in the MED group (0%).

Conclusion(s): The 2-cell block was specifically rescued by the microinjection of MPF and not by the insertion of pipettes.     

Peritoneal sperm recovery can be consistently demonstrated in women with unexplained infertility

Diligent analysis of PF 10 to 20 hours after midcycle intracervical insemination with husband's semen in couples with unexplained infertility showed that sperm are consistently able to transverse the reproductive tract in this group of patients. However, this finding does not necessarily imply that the sperm were retained at the site of fertilization or that they were competent to achieve oocyte fertilization. Therefore, further experiments obtaining sperm from the tubal isthmus to assess the effects of their sequestration there on their ability to fertilize human oocytes are needed.     

Sperm function in patients with unexplained infertility

Summary. Sperm function was studied in 27 patients with hitherto unexplained infertility. The ability of spermatozoa to reach the site of fertilization was assessed by laparoscopic sperm recovery from the peritoneal fluid and fimbrial rinsings and sperm fertilizing capacity with the zona-free hamster egg penetration in vitro test. The ability of spermatozoa to reach the site of fertilization correlated significantly with their fertilizing capacity in vitro , but was totally unrelated to any of the conventional criteria of semen quality, including the postcapacitation movement characteristics of the spermatozoa. Among patients with unexplained infertility, there are individuals with defects of sperm function which cannot be identified by conventional clinical techniques.     

The use of amniotic fluid and serum with propanediol in freezing of murine 2-cell embryos

Human and mouse embryos have been cultured in amniotic fluid (AF). Human AF and human serum (HS) are used in the freeze-thaw of 2-cell mouse embryos. Two hundred seventy-five 2-cell embryos were collected into phosphate-buffered saline with 20% HS and 20% AF and into 100% HS and AF. The embryos were cooled with propanediol as cryoprotectant at a controlled rate. After thaw, they were cultured in T6 with 3 mg/ml bovine serum albumin. Blastocyst formation post-thaw was 56/79, 44/70, 51/61, and 56/79 of intact embryos from 20% HS, 20% AF, pure HS, and pure AF (NS). But blastocyst hatching was better from embryos frozen in pure HS (22/61, compared with 16/79 for 20% HS; P less than 0.05). Hence there is no advantage in using AF in freeze-thaw, but pure HS may be of use.     

Closed-system solid surface vitrification versus slow programmable freezing of mouse 2-cell embryos

Purpose  To compare closed-system solid surface vitrification with slow freezing. Methods  Mouse 2-cell embryos (n = 348) were divided into vitrification, slow freezing and non-frozen groups. For vitrification, embryos were exposed to 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 10 min, then transferred into 17.5% EG, 17.5% DMSO, 0.25 M trehalose and 10% FBS in PBS. They were placed on hemi-straws and inserted into 0.5 ml straws inside a previously cooled aluminum cylinder. Slow freezing was done in straws by the conventional method. Results  Vitrified embryos had significantly higher survival, further cleavage and blastocyst formation rates than those in the slow freezing group (p < 0.001) and were comparable to controls. Blastocysts in the vitrification and control groups had significantly more cells than those in the slow freezing group (p < 0.05). Conclusions  Closed-system vitrification was more effective than conventional slow freezing. Capsule   Closed system solid surface vitrification was more effective than conventional slow freezing in the cryopreservation of mouse 2-cell embryos.     

Cryopreservation of mouse 2-cell embryos and ova by vitrification: methodologic studies

Cryopreservation of unfertilized mouse ova and 2-cell embryos by a vitrification technique was examined. Survival was defined by development to the hatching blastocyst stage after in vitro fertilization. With 19 embryos at the 2-cell stage, the authors obtained 100% morphologic survival and 89% development to hatching blastocyst stage. To define the optimal conditions for vitrification of ova, the authors treated a total of 845 unfertilized ova. In experiments done at 0 degree C, the concentration of vitrification solution (VS1) and the length of exposure of ova to VS1 both had significant (P less than 0.01) effects on survival. The mean survival rate for controls in ten experiments was 52%. VS1 100% or 90% in HEPES buffered saline and 10 minutes' exposure yielded rates that did not differ significantly from controls. Significantly lower survival rates followed the use of 70 and 80% solution and exposure for 5, 15, 20, or 30 minutes. Thus, under these conditions, exposure of unfertilized mouse ova to VS1 and cooling to 0 degree C did not interfere with in vitro fertilization and development of embryos. However, in five experiments in which a total of 101 ova were plunged into liquid nitrogen after treatment with VS1 under the optimal conditions, none could be fertilized in vitro.     

Peritoneal fluid and serum leptin concentrations in women with primary infertility

Aim  Leptin is proposed to participate in the reproductive system of women by acting on either ovaries or hypothalamic-pituitary axis. The objective of the present study is to investigate the leptin concentrations in peritoneal fluid and serum samples of women diagnosed with primary infertility. Methods  A prospective study was carried out in women who underwent laparoscopy within the diagnostic process of primary infertility between January 2005 and January 2007. Leptin concentrations were determined in blood samples obtained before surgery and in peritoneal fluid samples collected during laparoscopy. Results  Peritoneal fluid was obtained from 112 subjects; 21 with unexplained infertility 28 with polycystic ovary syndrome (PCOS), 30 with bilateral tubal occlusion, and 33 with endometriosis. Subjects with PCOS have significantly higher body weights, BMI values and plasma leptin levels when compared to other study groups. Peritoneal fluid levels of leptin were significantly higher in the endometriosis group compared to other three study groups. A positive correlation was found between peritoneal fluid leptin levels and the endometriosis stage (r = 0.51, P = 0.01). However, plasma leptin levels were unrelated to the disease extent. Discussion  It might be hypothesized that leptin may be an active factor in the pathogenesis of PCOS and endometriosis, which are two major causes of primary infertility. A mild leptin deficiency in peritoneal environment may interrupt follicular development and ultimately lead to PCOS. Leptin has angiogenic and mitogenic properties, which trigger inflammatory cytokines and eventually result in the development of endometriosis implants. Significantly, higher levels of leptin in peritoneal environments of endometriosis subjects strongly imply the important role of this common pathology.     

Peritoneal and peripheral B-1-cell populations in patients with endometriosis

OBJECTIVE: The purpose of this study was to investigate the frequency of B-1 cells in the peritoneal cavity and peripheral blood of patients with endometriosis. MATERIALS AND METHODS: We examined 31 patients with endometriosis and 14 normal nonpregnant women. Peripheral blood cells and peritoneal exudate cells (PECs) were stained with FITC or PE-labeled anti-CD5/CD19 monoclonal antibodies. Immunofluorescence analysis was performed using a flow cytometer. The significance of differences between the patient and control groups was determined by the non-parametric Mann-Whitney test. RESULTS: There was no significant difference in the percentages of B-1 cells in the peripheral blood of women with and without endometriosis (median, 22.7%; range, 4.7-92.3% vs median, 20.05%; range, 11.1-12.6%, respectively). Endometriosis patients with antinuclear antibodies (ANAs) demonstrated significantly elevated B-1 cells compared to both endometriosis patients without ANAs and normal controls (p < 0.005 and p < 0.05, respectively). Endometriosis patients demonstrated significantly higher B-1 cell populations (B-1 cells/total B-cell ratio) in PECs than did non-endometriosis patients (p < 0.05). CONCLUSIONS: The peripheral B-1-cell population in patients with endometriosis is related to ANA production. B-1 cells might play important roles in the development of endometriosis through autoantibody production.     

Present concept of unexplained infertility.

Despite improvements in both diagnostic assessment and treatment of infertile couples, many couples still have no explanation for their infertility. Unexplained infertility (the failure to conceive of a couple in whom no definitive cause for infertility can be found) has an incidence of 10-20% in all infertile couples. The incidence varies with the population studied and with the criteria used. Unexplained infertility is not an absolute condition but rather a relative inability to conceive, and many of these couples may conceive without treatment. The treatment options for unexplained infertility are several and the treatment results are promising. Expectant management can be recommended if the woman is under 28-30 years of age and the infertility duration is less than 2-3 years. In vitro fertilization (IVF) has revolutionized the treatment of infertile couples, as well as profoundly increasing the basic understanding of human reproduction. IVF can be used as both a diagnostic and a therapeutic tool in couples with unexplained infertility. The pregnancy rates with IVF are good, at 40% per treatment cycle. In addition, the outcome of pregnancies among women with unexplained infertility is generally comparable to that of spontaneous and other pregnancies using assisted reproductive technologies.