Human motile sperm recovery after cryopreservation: freezing in nitrogen vapor vs the direct plunge technique.

Objectives: The following will be tested: 1) Freezing in nitrogen vapor prior to submerging will have a higher motile sperm recovery rate compared to directly submerging in liquid nitrogen, 2) The length of time a sample spends in nitrogen vapor will not affect the motile sperm recovery, and 3) The volume of sample to be frozen will not affect the recovery of motile sperm.Design: This is a prospective study performed through a university infertility clinic. The semen samples were obtained from 20 male partners of couples being screened for in vitro fertilization.Materials and Methods: Semen was collected and washed with 5 volumes of modified Ham's F10 medium and centrifuged at 120xg for 10 minutes. The pellet was resuspended in 5 mL of medium and centrifuged in a similar manner. The swim-up sperm was prepared with the second pellet overlaid with 1 mL of medium, containing 1% human serum albumin, and incubated for 1 hour at 37 C. Equal volumes of sperm suspension and cryoprotectant were mixed by gentle stirring. Sperm density and motility were recorded before freezing. Each vial contained 1 mL of the sperm/cryoprotectant mixture. All vials were eventually submerged in liquid nitrogen for 40 minutes and thawed at room temperature for 1 hour. The percent of motile sperm recovered was assessed. Hypothesis 1: Comparing freezing in nitrogen vapor versus direct plunge, one vial was frozen for 6 minutes in the nitrogen vapor 2 cm above the liquid surface prior to submerging. The paired vial was kept at room temperature for 6 minutes before submerging. Hypothesis 2: Two vials were frozen in vapor for 6 and 12 minutes prior to submerging them. Hypothesis 3: To assess the effect of sample volume on motile sperm recovery, three separate vials were prepared, each containing either 1.0, 0.5, or 0.25 mL of the sperm/cryoprotectant mixture. Each vial was suspended in vapor for 6 minutes prior to submerging into the liquid. Paired t test analysis was used for objectives 1 and 2, with repeated measures analysis of variance used to determine the significance of objective 3.Results: Pure motile sperm density and motility were assessed for 20 samples after the addition of cryoprotectant. The sperm density and motility ranged from 0.9 to 120 million sperm/mL (33.2, SD +/- 35.0) and 78-100% (86.5, SD +/- 12.4), respectively. The mean motile sperm recovered for vapor freezing prior to plunge was 54.9% (SD +/- 15.4), compared to submerging directly, which was 21.5% (SD +/- 10.0); this was significant with a P <.05. The initial sperm density, <20 or >/=20 million/mL, did not influence the percent of motile sperm recovered (P >.05). Motile sperm recovered from samples in vapor for 6 and 12 minutes was 49.7% (SD +/- 8.71) and 51.8% (SD +/- 10.4), respectively; this was not significant. The volume of purified motile sperm frozen did not significantly alter the percent of motile sperm recovered.Conclusion: Directly submerging a sample from room temperature into liquid nitrogen has shown a reduction of motile sperm recovery by 65.0%, compared to exposing the sample to liquid nitrogen vapor prior to submerging, which only showed a reduction of 31.7%. Motility appears to be influenced by the rate at which the sample is frozen. There was no significant impact on motile sperm recovery compared to the amount of sperm preserved, length of time the sample was in liquid nitrogen vapor, or the initial sperm density. This is the first study to isolate the motile faction from semen and examine the recovery rate between the two cryopreservation techniques. The clinical application implies that increasing the recovery of motile sperm could improve pregnancy rates and this simplified technique is possible without obtaining special equipment that could impose a significant financial burden with similar results.

Serum and bronchoalveolar lavage of N-terminal type III procollagen peptides in idiopathic pulmonary fibrosis.

Collagen deposition is a prime determinant of clinical course in idiopathic pulmonary fibrosis (IPF). Identification of a marker of connective tissue metabolism would significantly enhance the ability to stage the disease and monitor the course of these patients. Prior studies of IPF have indicated that N-terminal Type III procollagen peptide (N-PIIIP) levels in blood and bronchoalveolar lavage BAL fluid are elevated. We hypothesized that elevated levels of procollagen peptides are a marker of enhanced collagen deposition, which is associated with interstitial fibrosis characterizing active disease. The purpose of the present study was to explore the relationship between N-PIIIP recovery and physiologic parameters of lung function. N-PIIIP levels in sera and bronchoalveolar lavage (BAL) from 24 patients with IPF and 29 volunteers were measured by radioimmunoassay. The extent of disease in IPF was assessed by clinical history, physical examination, chest radiograph, pulmonary physiology evaluation, and confirmatory open-lung biopsy. The severity of disease was graded using a previously described clinical, radiologic, and physiologic (CRP) scoring system. N-PIIIP normalized to albumin was higher in BAL than in serum for both volunteers (1.6-fold; p less than 0.05) and IPF patients (24-fold; p less than 0.05), consistent with local pulmonary production. BAL N-PIIIP was significantly elevated in IPF patients, whether expressed as concentration (healthy volunteer 0.11 +/- 0.06 ng/ml; IPF, 5.0 +/- 14.4; mean +/- SD; p less than 0.05) or normalized to albumin (healthy volunteer, 2.8 +/- 1.2; IPF, 73 +/- 106; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

CA-125 levels in human uterine fluid.

OBJECTIVE: To measure uterine fluid CA-125 concentration and to determine if any menstrual cycle phase dependent changes exist in its level. Serum levels are measured for comparison. DESIGN: CA-125 levels in uterine fluid were measured during the follicular and luteal phases of the menstrual cycle. In a sequential study, paired uterine fluid and serum samples were obtained once in both midfollicular and midluteal phases of the same menstrual cycle. RESULTS: CA-125 in uterine fluid during the follicular phase (n = 14) ranged from 16.4 x 10(3) to 616.5 x 10(3) U/mL, and from 6.2 x 10(3) to 567.3 x 10(3) U/mL in the luteal phase (n = 11). In the paired sequential uterine fluid and serum samples, (1) the means (+/- SEM) CA-125 in uterine fluid were 81.5 x 10(3) +/- 37.9 x 10(3) U/mL and 91.4 x 10(3) +/- 56.8 x 10(3) U/mL in the midfollicular and midluteal phases, respectively (P = 0.75); (2) the CA-125 levels in serum increased in the midluteal phase (P less than 0.05); and (3) compared with serum, uterine fluid levels were greater with a wider range. CONCLUSIONS: When compared with serum CA-125, uterine fluid contains high concentrations varying over a wide range without fluctuation between the follicular and luteal phases of the menstrual cycle.

Prostaglandin F2 alpha synthesis and metabolism by luteal phase endometrium in vitro.

Differences in prostaglandin (PG) F2 alpha synthesis and degradation were sought between early luteal endometrium (histology day 15 to 22, n = 6) and late luteal endometrium (histology day 23 to 28, n = 6). In addition, alterations in PGF2 alpha synthesis and degradation in response to 17 beta-estradiol (E2) and progesterone (P) were examined to clarify the mechanism of steroid modulation of endometrial PG production (n = 12). Endometrium was maintained in tissue culture and the concentration of PGF2 alpha and 13,14 dehydro-15 keto F2 alpha (DHKF2 alpha) was determined in spent media by radioimmunoassay. Prostaglandin F2 alpha and DHKF2 alpha output from luteal endometrium exposed to P and E2 + P were both significantly reduced when compared with no addition or E2 treatments. This implies that the modulation of PGF2 alpha output by P in vitro is secondary to altered synthesis. There was an increase in PGF2 alpha output from late luteal endometrium when compared with early luteal endometrium in the P and E2 + P treatments, but DHKF2 alpha remained unchanged. These data imply that the temporal increase in PGF2 alpha output is the result of alterations in both PG synthesis and catabolism.

Observation of production of immunoactive prolactin by normal human connective tissue in cell culture.

Data from our in vitro studies indicate a new source of prolactin (PRL)-like activity, normal human connective tissue. Fascial cells from primary culture and subsequent passages produced an extracellular antigen which specifically reacted in a radioimmunoassay RIA developed to detect human pituitary PRL. An initial peak or first surge of fascial PRL-like activity occurred between 4 and 15 d in primary culture. Ibuprofen, cytotoxic levels of 0.01% azide, or 7.5 mM EDTA and medium lacking serum [fetal bovine serum (FBS)] significantly (P less than or equal to 0.05) reduced PRL-like activity levels, whereas female steroids, 257 to 342 milliosmolarity, 1 to 3.6 mg/ml glucose, 2 to 20% FBS, and dialyzed FBS (MWCO approximately 1 kDa) were without effect. Optimum production of PRL-like activity occurred at pH 7.3. A second surge began after 18 d and continued until passage indicating that perhaps two populations of cells produced PRL-like activity in primary culture. Production of PRL-like activity by cells from early passages (1 and 2) became detectable at confluence, was serum-dependent, showed two patterns (tonic, rising to plateau), and averaged 3.2 fg.cell-1.3 d-1 feed interval. Cells from late passages showed morphologic damage from repetitive trypsinization, aging, and reduced production of PRL-like activity with aberrant production pattern. Production of PRL-like activity was maintained in an unusual long-term culture. These in vitro studies demonstrate the most recently recognized and ubiquitous source of human extrapituitary PRL or PRL-like activity, normal connective tissue (fascia).

Physicochemical characterization and functional activity of fibroid prolactin produced in cell culture.

Evidence from our laboratory with the use of cultured (primary and passaged) cells has extended our initial observation that human uterine fibroid is an extrapituitary source of prolactin. Fibroid prolactin antigen in conditioned medium reacted specifically in radioimmunoassay for human pituitary prolactin. Control experiments demonstrated that the radioimmunoassay results were not spurious due to degradation of tracer 125I-labeled prolactin. Immunoparallel dilution curves indicated antigenic relatedness of pituitary and fibroid prolactin. In a calibrated Sephadex G-100 column, fibroid prolactin eluted in the same region (20.3 to 20.9 kd) as purified pituitary prolactin. Glycosylated prolactin, detected by concanavalin A affinity column chromatography, appeared to constitute only a small percentage of fibroid prolactin made in culture. The ratio of fibroid prolactin bioactivity (lactogen Nb2 lymphoma bioassay) to antigen (radioimmunoassay) was 0.77. These data indicate that human uterine fibroid tissue produces a molecule similar to or, perhaps, identical with pituitary prolactin.

Norethindrone inhibition of testosterone secretion by an ovarian Sertoli-Leydig cell tumor.

A woman with severe hyperandrogenemia and virilization was found to have a fall in serum testosterone (T) concentration while taking an oral contraceptive containing norethindrone (500 vs. 164 ng/dL). Subsequent surgical exploration revealed an ovarian Sertoli-Leydig cell tumor. In vitro, the tumor secreted T (mean, 1.88 ng/mg X 4 h). hCG did not stimulate T secretion. In addition, norethindrone inhibited T secretion (0.33 ng/mg X 4 h). We conclude that norethindrone directly suppressed T production by the Sertoli-Leydig cell tumor.

Uterine fluid and prolactin secretion in the ovulating cynomolgus monkey.

Immunoreactive prolactin is produced by late secretory human endometrium in vitro. Human myometrium in explant culture also produces prolactin. A primate model with the use of the cynomolgus monkey is described that allowed repeated samplings of uterine secretions in vivo. The uterine secretory prolactin thus measured appears immunoreactively similar to human serum prolactin, and the pattern of secretions reflects the previously described pattern of endometrial prolactin production in vitro.

Prolactin production from proliferative phase leiomyoma.

In vivo and in vitro endometrial stromal synthesis of prolactin occurs after progesterone-induced decidualization. Synthesis of prolactin by myometrium in vitro suggests that cells whose embryologic origin is the loose mesenchyme surrounding the paramesonephric ducts may retain the capacity to synthesize prolactin. Since physiologic myometrial synthesis of prolactin has not been demonstrated in vivo, prolactin genome expression in pathologic conditions was considered. Follicular phase leiomyomas were diced to 8 mm3 and cultured in Dulbecco's modified Eagle's medium (DMEM) with either no hormones, estradiol 200 pg/ml, progesterone 20 ng/ml, or estradiol and progesterone. Media were sampled and changed every other day for 8 days, followed by culture in tritium-labeled leucine DMEM for 2 days. Portions of leiomyomas were homogenized for initial prolactin content, and all samples were assayed for prolactin by radioimmunoassay. Follicular phase leiomyomas contained prolactin (47 +/- 15 ng/gm) in excess of normal serum values. Synthesis was demonstrated during all time periods from leiomyomas not exposed to progesterone. Progesterone variably suppressed the synthesis of prolactin until after 144 hours of culture. Determination of molecular weight on a 60 by 1.5 cm Sephadex G-100 column revealed identical estimates for pituitary, decidual, and leiomyoma prolactin. Tritium-labeled leucine incorporation into prolactin was confirmed by immunoprecipitation of Sephadex G-100 column fractions. Similar antigenicity was confirmed by parallel dilution curves for pituitary, decidual, and leiomyoma prolactin. Preliminary bioactivity in lymphoma proliferation assays confirmed prolactin activity. The conclusion reached was that proliferative phase leiomyomas contained elevated prolactin presumably secondary to in vivo synthesis. This synthesis was confirmed in vitro.

Human myometrium: a new potential source of prolactin.

Human myometrium is shown for the first time to produce prolactin in vitro. This prolactin is similar to pituitary prolactin by criteria of immunologic identity, gel chromatography and bioassay. The de novo synthesis of myometrical prolactin is supported by no detectable prolactin in initial tissue homogenate, nondetectable prolactin production during the first 24 hours of culture, cycloheximide inhibition of prolactin production with recovery of production in control medium, and tritiated leucine incorporation into prolactin. Although human myometrium is capable of producing prolactin without the addition of exogenous hormones, the addition of estrogen and progesterone, respectively, enhances and suppresses prolactin production in contrast to decidualized human endometrium where opposite effects on prolactin production are found.

Familial partial deficiency of the third component of complement (C3) and the hypocomplementemic cutaneous vasculitis syndrome.

Familial hypocomplementemia of the third component of complement (C3) was found in four members of a family. The prospositus had cutaneous vasculitis, hypocomplementemia, arthralgia, proteinuria and thrombocytopenia. The combination of clinical, laboratory and pathologic findings resembled the "hypocomplementemic cutaneous vasculitis syndrome" (HCVS) or the "SLE-like syndrome" but serum C3 concentration was 35 to 57 per cent of normal in the propositus and in three relatives. Results of Clq precipitins, cryoglobulins and serologic tests for systemic lupus erythematosus were negative. Proteinuria (815 mg/day) but no hematuria was present. Analysis of the C3 phenotypes in this family showed that three hypocomplementemic members were apparent homozygous C3 slow but one was heterozygous C3 fast-slow. Metabolic studies with 125-Iodinated C3 in the clinically normal mother showed a 50 per cent reduction in C3 synthesis which was consistent with hypocomplementemia documented by serum protein assay. The occurrence of an immune complex-like disease (with characteristics of the HCVS) in a patient with a familial deficiency of C3 suggests that the preexisting C3 deficiency may predispose such persons to certain diseases.

Multiple sedimenting species of properdin in human serum and interaction of purified properdin with the third component of complement.

Normal human serum subjected to sucrose density gradient analysis exhibited multiple sedimenting species of properdin antigen. Properdin antigen distribution was dependent on serum concentration, ionic strength, temperature, and the presence of C3, and was not dependent on the presence of divalent metal cations or blood coagulation. In mixtures of purified components, properdin sedimented heavier in the presence of C3, C3b, or C3c. Addition of factor B to mixtures containing C3 and properdin was without effect. These data provide insights into earlier discrepancies concerning the sedimentation behavior of partially purified properdin, indicate a propensity of some constituents of the alternative pathway to form protein-protein complexes, and suggest caution in interpretation of immunopathological studies in which properdin deposits are found in the presence of C3.