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Biomedical subjects

D P Wolf

Publications and source records attributed to D P Wolf.

At least 19 recordsLinked to original sources
PubMed1992

Cortical granule complements in human oocytes undergoing partial zona dissection.

This study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P less than 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.

Cells, Cultured↗
PubMed1992

Intrauterine insemination outperforms intracervical insemination in a randomized, controlled study with frozen, donor semen.

OBJECTIVE: To assess the efficacy of intrauterine insemination (IUI) in a donor insemination program. DESIGN: Prospective randomized clinical trial. SETTING: Donor insemination program. PATIENTS, PARTICIPANTS: Women undergoing insemination were randomly assigned to receive either IUI or intracervical insemination for a maximum of six cycles. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Cycle fecundity rates between the two routes were compared. RESULTS: The monthly fecundity rate for intracervical insemination was 5.1% compared with 23% by IUI. By life table analysis, pregnancy rates for IUI were significantly higher than intracervical insemination (P = 0.02). CONCLUSIONS: Intrauterine insemination with quarantined donor sperm is superior to intracervical insemination.

Cervix Uteri↗
PubMed1992

The Sulawesi Crested Black Macaque (Macaca nigra) menstrual cycle: changes in perineal tumescence and serum estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone levels.

Events in the normal menstrual cycle of the endangered Sulawesi Crested Black Macaque (Macaca nigra) were characterized. Daily blood samples were obtained during 10 menstrual cycles from five M. nigra demonstrating regular cycles. The amount of perineal tumescence was scored daily. Serum levels of estradiol and progesterone were determined by RIA, serum LH levels were determined by the mouse Leydig cell bioassay, and serum FSH levels were determined by the rat granulosa cell aromatase bioassay. Cycle length was 39.8 +/- 1.0 days (mean +/- SEM) with an LH surge occurring 25 +/- 1.5 days from the onset of menses. After menses, both LH and estradiol were initially depressed, with estradiol first exceeding 50 pg/ml 8 days before the LH surge. In five cycles, peak estradiol levels (340 +/- 44 pg/ml) occurred on the day of the LH surge (637 +/- 58 ng/ml) and in the other five cycles, on the day before the LH surge. There was a broad increase of FSH in midcycle without a well-defined surge corresponding to the LH surge. Progesterone began increasing on the day of the LH surge and reached peak levels (6.8 +/- 0.96 ng/ml) 8 days later. Maximal perineal tumescence was generally associated with the time of the LH surge, but variation between animals made it impossible to predict accurately the day of the LH surge by perineal tumescence scores alone.

Animals↗
PubMed1992

Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles.

After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Successful hysteroscopic cannulation and tubal transfer of cryopreserved embryos.

Clinical trials comparing nonsurgical transfer techniques with laparoscopic directed methods are needed to determine the most successful and cost-effective approach for gamete and ET. We report the successful nonsurgical transfer of frozen-thawed embryos into the fallopian tube after our initial attempt resulting in an ongoing IUP. The process is relatively simple, safe, and avoids the risks of general anesthesia. Unlike US-guided tubal cannulation, this technique offers direct, clear visualization of the tubal ostium, assuring the desired catheter placement. As well, hysteroscopic guidance allows an accurate estimate of the depth of catheter insertion within the fallopian tube, which may be a critical factor in successful tubal deposition of gametes or embryos. Whether prolonged carbon dioxide exposure of the tubal microenvironment and/or direct endometrial trauma limits the efficacy of this technique remains to be determined. Finally, outpatient hysteroscopic directed tubal cannulation holds promise as a methodological technique that ultimately assists in the examination of the best site for embryo deposition (tubal versus uterine) and/or method of delivery (nonsurgical versus surgical).

Adult↗
PubMed1991

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. I. Oocyte maturation and corpus luteum function.

The amplitude and duration of the midcycle LH surge required for ovulatory maturation of the follicle and its enclosed oocyte in primates are unknown. To titrate periovulatory LH requirements, female rhesus monkeys received human gonadotropins (FSH with/without LH) for 9 days beginning at menses to promote the development of multiple preovulatory follicles. The next day, animals (n = 4-6/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h) to induce ovulatory maturation. Follicles were aspirated 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 and 10 in animals receiving no ovulatory stimulus. Nuclear maturity of oocytes was evaluated as a marker for reinitiation of meiosis. Estradiol and progesterone levels were determined in daily serum samples by RIA. Levels of LH(-like) bioactivity were measured at selected intervals after hCG injection and within 24 h of GnRH/GnRHa treatment. In all groups, estradiol continuously rose to similar peak levels on day 10. The hCG treatment markedly elevated circulating LH-like bioactivity for up to 3 days. In GnRH-1, bioactive LH increased to 433.1 +/- 170.2 ng/mL (mean +/- SEM; n = 3) within 1-2 h, but then decreased to baseline (4.9 +/- 1.5 ng/mL) within 6 h. GnRH-3 and GnRHa treatment extended the interval of elevated bioactive LH to 8 and 14 h, respectively. There was no difference in the peak levels of LH(-like) bioactivity reached after hCG, GnRH, or GnRHa injection. Functional luteal phases were absent in monkeys receiving no ovulatory stimulus, whereas hCG treatment increased progesterone levels to 101 +/- 9 nmol/L (n = 6) and elicited functional luteal phases of 11.8 +/- 0.4 days. In contrast, only one animal in the GnRH/GnRHa groups (i.e. one GnRH-3 monkey) displayed elevated progesterone levels in the luteal phase. Of the total cohort of oocytes aspirated from follicles, a greater (P less than 0.05) proportion were classified as being in metaphase I or II of meiosis after hCG treatment (86%) compared to no ovulatory stimulus (13%), GnRH-1 (0%), GnRH-3 (43%), and GnRHa (12%). Thus, GnRH elicits a transient LH surge that can be extended by GnRH-3 or GnRHa in stimulated cycles of monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Collection and quality of rhesus monkey semen.

Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electroejaculation: 1) a constant-voltage method where stimulus current is a variable and 2) a constant-current method where stimulus current is operator-controlled. The constant-current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The influence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were detrimental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra- and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.

Animals↗
PubMed1990

The use of gonadotropin-releasing hormone agonist to regulate oocyte retrieval time.

We investigated the use of a gonadotropin-releasing hormone agonist (GnRH-a) to assist in the synchronous entry of a fixed number of subjects into in vitro fertilization (IVF) and gamete intrafallopian tube transfer cycle. Using a protocol in which a GnRH-a was given in the follicular or luteal phase, the clinical pregnancy rate per procedure was 27%. We conclude that the use of a GnRH-a for synchronous cycles in IVF-embryo transfer is efficacious and results in acceptable pregnancy rates.

Embryo Transfer↗
PubMed1990

A comparative evaluation of intracervical and intrauterine routes in donor therapeutic insemination.

The relative efficiency of intrauterine insemination with washed spermatozoa versus intracervical deposition of semen was examined in donor insemination for therapeutic reasons. Fecundability in a group of 26 women who desired insemination as a treatment for azoospermia or severe astheno-oligozoospermia in their partner was comparable, despite the 10-fold higher numbers employed using the intracervical route.

Cervix Uteri↗
PubMed1990

Maturity at collection and the developmental potential of rhesus monkey oocytes.

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions.

The kinetics of spontaneous and induced acrosomal loss have been studied in human sperm incubated in capacitating and noncapacitating media. Acrosomal status was quantitated using indirect immunofluorescence with a monoclonal antibody. The response of sperm to induction by calcium ionophores was time dependent reaching a maximum after 6 hours of incubation under capacitating conditions. The inducible population slowly decreased in size through the balance of a 24-hour incubation. The time-dependent development of ionophore responsiveness by sperm exposed to capacitating conditions corroborates the idea that only capacitated cells can respond to undergo acrosomal loss in response to ionophore. In contrast, only a small, constant percentage of sperm incubated under noncapacitating conditions responded to ionophore. Substitution experiments involving the addition or deletion of human serum albumin suggest that albumin is not absolutely required for capacitation but is essential for the maintenance of motility. Polyvinyl alcohol can be substituted for serum albumin, but it does not support capacitation or motility as well as HSA. These studies may provide a basis for optimizing capacitating conditions for human sperm in vitro as well as for diagnosing fertility or fertility potential based on measurements of spontaneous and ionophore induced acrosomal loss under defined culture conditions.

Acrosome↗
PubMed1989

Human sperm capacitation in gelatin-fortified medium.

Gelatin was shown to be an effective substitute for serum albumin in human sperm capacitation. No protein-dependent differences were seen with regard to long-term retention of sperm motility, including hyperactivation, in acrosomal status or in the acquisition of fertility potential. Gelatin may serve as a cost-effective substitute for serum albumin, which carries minimal risk of infectious disease transmission.

Culture Media↗
PubMed1989

Effect of measurement conditions on quantification of hyperactivated human sperm subpopulations by digital image analysis.

The effect of different measurement conditions on the quantification of hyperactivated (HA) motility in human sperm by digital image analysis (DIA) was determined with respect to chamber depth, temperature, sperm concentration, time in the analysis chamber, culture medium, and type as well as concentration of extracellular protein components. In whole semen, the level of HA (less than 1%) as well as the values for all other motility parameters (curvilinear and straight-line velocity, linearity of forward progression, maximum and mean lateral head amplitude, and beat cross frequency) were independent (p greater than 0.05) of temperature (24 vs. 37 degrees C). In washed sperm samples prepared by in vitro fertilization procedures, the percentage of HA cells was inversely related to measurement temperature and to albumin concentration in the culture medium. The effect of temperature on percentage of HA in washed sperm was reversible. HA analysis of washed sperm from 6 donors (3 ejaculates per donor) under defined DIA assay conditions showed no intra-donor variability (p greater than 0.05) but significant inter-donor variability (donor mean percentages ranging from a low of 3.6 to a high of 28.2). These results indicate that HA is influenced by measurement conditions, that HA is donor-dependent, and that each donor shows a characteristic level of HA.

Culture Media↗
PubMed1989

In vitro fertilization and embryo transfer in the rhesus monkey.

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.

Animals↗
PubMed1989

Acrosomal status quantitation in human sperm.

Acceptable methods are currently available for the rapid quantitation of acrosomal status in human sperm. Application of these methods to human sperm has led to the concept that spontaneous acrosomal loss occurs infrequently during capacitation. Induced loss occurs following sperm exposure to human follicular fluid, to zonae- or cumulus-enclosed eggs, or to calcium ionophores or other agents that modulate intracellular calcium levels. Acrosomal status quantitation may be helpful in sperm morphologic characterization or in evaluating cryodamage but has not yet been established in fertility potential measurement.

Acrosome↗