Pregnancy rates in a donor sperm program using cryopreservation: effects of total number of motile sperm inseminated and of a procedure to concentrate sperm.

OBJECTIVE: To determine the relationship between the total number of motile sperm per insemination (TMSI) and pregnancy rates in a donor insemination program. To determine the effect of sperm concentration and resultant increase of TMSI on pregnancy rates. DESIGN: Retrospective analysis of pregnancy rates. SETTING: University hospital with tertiary service. PATIENTS: 179 women undergoing donor insemination. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Calculation of pregnancy rates according to TMSI. RESULTS: The pregnancy rate with insemination of thawed donor sperm increases when the TMSI of unconcentrated sperm is > or = 80 x 10(6). Pregnancy rates with previously concentrated sperm are as good as or better than rates of unconcentrated specimens. CONCLUSION: TSMI is important in determining pregnancy rates in donor insemination programs, and concentration of sperm prior to freezing may enhance pregnancy rates by increasing the TMSI.

Temporal relationship and reliability of the clinical, hormonal, and ultrasonographic indices of ovulation in infertile women.

To assess ovulatory function, 50 infertile but "normally" ovulating women were monitored closely during a single menstrual cycle with daily measurements of serum estradiol, progesterone, and LH (morning and evening urinary LH by standard radioimmunoassay and morning urinary LH by enzyme-linked immunosorbent assay); transvaginal ultrasound; basal body temperature (BBT) recording; and cervical mucus. All 50 cycles, 40 spontaneous and ten induced with clomiphene citrate, were ovulatory. Follicle rupture was confirmed by ultrasound in 47 cycles (94%), whereas three patients fulfilled the diagnostic criteria for luteinized unruptured follicle syndrome. Follicle rupture occurred on day +1 in three, on day +2 in 34, and on day +3 in ten of the ovulatory cycles. Urine LH testing correlated well with the serum LH peak, particularly in the evening urine, and predicted ovulation in all patients. The initial periovulatory rise in serum progesterone occurred on the same day as the LH surge (day 0) in 30%, on day +1 in 40%, on day +2 in 22%, and on day +3 or later in 8% of the cycles. The rise in BBT corresponded to an increase in serum progesterone to 5 ng/mL or greater. Neither the BBT nor cervical mucus was reliable in predicting ovulation.

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).

Stimulation of uterine prolactin secretion by human chorionic gonadotropin and progesterone in the cynomolgus monkey.

The ovulating cynomolgus monkey secretes immunoreactive prolactin (PRL) into the uterine cavity. Consistent with human endometrial explant data, uterine PRL is undetectable in the early secretory phase, then increases from the mid to late secretory phase, peaking premenstrually. The anovulatory monkey does not produce detectable uterine PRL. Human chorionic gonadotropin given repeatedly fails to induce PRL secretion in anovulatory monkeys but prolongs the luteal phase and thereby PRL secretion in ovulatory monkeys. Progesterone (P) induces PRL secretion in anovulatory monkeys after estrogen priming with a time delay of several days, indicating probable de novo synthesis. P appears to be an important stimulating factor in the control of uterine PRL secretion.

Human uterine luminal fluid volumes and prolactin levels in normal menstrual cycles.

Human uterine luminal fluid has not been well characterized. Prolactin is produced in vitro by decidualized human endometrium and is secreted in vivo into the uterine luminal fluid of cynomolgus monkeys. Uterine luminal fluid prolactin has not been demonstrated in vivo in human beings. To study cyclic uterine luminal fluid volumes and prolactin levels, uterine luminal fluid was aspirated 8 to 13 times each during a single menstrual cycle in six ovulatory women. No anesthesia was used. Seventy of 75 (93%) attempts were successful; there were no complications. Serum estradiol, progesterone, luteinizing hormone, and prolactin levels were assayed every 1 to 3 days. Uterine luminal fluid volume and prolactin were normalized to the luteinizing hormone peak (day 0). Uterine luminal fluid volumes were relatively constant until the early luteal phase, when they then decreased. Uterine luminal fluid prolactin was detectable in all samples. Mean values were stable until day +3 or +4, after which they rose to a peak at day +9 or +10. This study establishes that (1) frequent uterine luminal fluid sampling is possible; (2) uterine luminal fluid volume decreases in the luteal phase; (3) uterine luminal fluid prolactin is detectable in vivo in women and its concentration increases in the luteal phase; and (4) cyclic human uterine luminal fluid prolactin levels differ from those in cynomolgus monkeys and from those anticipated from human in vitro studies.

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.

Evidence of short-loop inhibition of decidual prolactin synthesis by decidual proteins. Part I.

Induction of uterine endometrial prolactin synthesis is dependent on progesterone-induced decidualization of stromal cells. These decidual cells are not dependent on progesterone for continued prolactin synthesis. The factors modifying decidual prolactin synthesis remain largely unknown. To test the hypothesis that a decidual protein is the major modulator of new prolactin synthesis, decidua were cultured within dialysis membranes allowing the accumulation of proteins greater than 12,000 molecular weight in a metabolically neutral environment, and the rate of new synthesis was compared with prolactin synthesis from samples cultured in 10 times the available volume for protein distribution. The rate of new prolactin synthesis at 48-hour intervals up to 144 hours was compared. Initial and postculture decidual prolactin content was obtained and was found not to vary significantly between groups (0.05 less than p less than 0.10). At 48 hours significant suppression of decidual prolactin synthesis was apparent (p less than 0.05) within the dialysis membranes. As prolactin concentration increased during in vitro culture this suppression was enhanced (p less than 0.005). Gel chromatography and immunoprecipitation of iodine 125-labeled prolactin added at time 0 revealed no significant degradation of the 125I-labeled prolactin and maintenance of its immunoactivity even at 144 hours. This confirms that the plateauing of prolactin concentration within the dialysis membranes is due to suppression of new synthesis rather than metabolism of previously synthesized prolactin.

Evidence of short-loop inhibition of decidual prolactin synthesis by decidual proteins. Part II.

We have recently demonstrated the suppression of decidual prolactin synthesis by decidual proteins accumulating in a dialysis membrane. In this report the reversibility of this suppression and the molecular weight range of the suppressor(s) are examined. Samples of 300 mg of decidua (n = 10) were cultured in three environments: in 30 ml of medium (control), in 3 ml of medium in a dialysis membrane suspended in 27 ml of medium, and in 2.7 ml of medium with 0.3 ml of concentrated decidual proteins of 10,000 to 35,000 molecular weight (giving a time 0 prolactin concentration of 220 ng/ml) in a dialysis membrane suspended in 27 ml of medium (n = 5) or in 2.7 ml of medium with 0.3 ml of concentrated decidual proteins greater than 35,000 molecular weight (giving a time 0 prolactin concentration of 5 ng/ml) in a dialysis membrane suspended in 27 ml of medium (n = 5). Dialysis membranes were discontinued at 48, 96, and 144 hours and the decidua within was subsequently cultured in 30 ml of medium until 240 hours. The suppression of prolactin synthesis identified previously was confirmed in this study. Further it was found that the suppression of decidual prolactin synthesis was reversed by the removal of decidua from the high decidual protein-prolactin environments of the dialysis membranes. Third, only the addition of decidual proteins between 10,000 and 35,000 molecular weight with a high prolactin concentration enhanced the suppression of new prolactin synthesis. We conclude that a decidual protein(s) between 10,000 and 35,000 molecular weight, possibly prolactin itself, is the primary control factor(s) in the modulation of prolactin synthesis in term decidua. Further the normal physiologic state of decidua is a high prolactin concentration in equilibrium with low prolactin synthesis. Therefore studies intended to assess the effects of modulators of prolactin synthesis in decidua should not be performed in low prolactin concentration-high prolactin synthesis conditions during short time periods (as has been the case in a majority of studies to date) since such conditions do not reflect normal physiologic conditions and may result in invalid conclusions.

Prolactin production by explants of normal, luteal phase defective, and corrected luteal phase defective late secretory endometrium.

The production of prolactin by explants of late secretory endometrium has been correlated with the extent of decidual differentiation. This correlation is strengthened by the observation that luteal phase defective endometrium produces less prolactin than normal control endometrium in a 24-hour in vitro culture system. In the present study the prolactin production by explants of normal, luteal phase defective, progesterone-corrected luteal phase defective, and clomiphene- or follicle-stimulating hormone/luteinizing hormone-corrected luteal phase defective late secretory endometrium was measured over 96 hours at 24-hour intervals. Progesterone in physiologic concentrations was added to the culture medium to maintain tissue integrity and prolactin synthesis. The prolactin production of normal late secretory endometrium rose over 96 hours under progesterone stimulation. The luteal phase defective endometrium produced significantly less prolactin under the same conditions. Histologically proven corrected luteal phase defective endometrium, regardless of treatment method, produced prolactin not different from the normal controls of the same dates. From these results it is concluded that histologic correction of luteal phase defective endometrium is associated with a corresponding biochemical correction with use of prolactin as a metabolic marker. The findings also strongly support timed endometrial biopsy as the method of diagnosis and evaluation of treatment of luteal phase defect.

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.