Diane G. Hammitt

Increased risk of monochorionic twinning associated with assisted reproduction.

OBJECTIVE To determine the incidence of monochorionic twinning in pregnancies resulting from assisted reproduction technologies (ARTs). METHODS We reviewed our experience with 218 ART pregnancies achieved over 3 years. All patients underwent transvaginal ultrasound 26 and 36 days after oocyte retrieval. The presence of two yolk sacs or two fetal poles within one sac suggested monochorionicity, and was confirmed by follow-up ultrasound and placental pathology. The various ARTs were compared to determine if any method had an increased incidence of monochorionicity compared to any other method. Statistical analysis was performed employing Chi Square analysis. RESULTS The incidence of monochorionicity in all gestations was 3.2% (8 times background rate); among multiple gestations it was 9.8%. The rates of monochorionicity for each ART appeared similar. CONCLUSION The incidence of monochorionic twinning is increased in pregnancies resulting from ART. Careful ultrasound evaluation of such pregnancies for monochorionicity is strongly recommended, both for planning of prenatal care and when considering a multifetal pregnancy reduction procedure.

Comparison of motility stimulants for cryopreserved human semen.

Caffeine, pentoxifylline, 2-deoxyadenosine, cyclic adenosine monophosphate (cAMP), relaxin, adenosine, kallikrein, and calcium were compared for their ability to stimulate motility of cryopreserved sperm. Caffeine, pentoxifylline, and 2-deoxyadenosine significantly increased the percentage of motile sperm at 15, 30, 45, and 60 minutes after administration. Sperm velocity was significantly increased by caffeine at 0, 15, 30, and 45 minutes, and by pentoxifylline at 0, 45, and 60 minutes. Consistent stimulation was not observed for other chemicals. Caffeine, pentoxifylline, and 2-deoxyadenosine were then examined for their ability to provide motility stimulation after removal with washing. With the exception of caffeine, percent motility and velocity for stimulated and untreated sperm were similar after washing. A significant reduction in motility was observed at 48 hours after washing for caffeine. The percentage of hamster oocytes penetrated at 24 hours after washing was significantly reduced for caffeine, 2-deoxyadenosine, and pentoxifylline combined with 2-deoxyadenosine. Pentoxifylline-treated sperm showed no reduction in fertilizing capacity. These results indicate that, of the chemicals examined, pentoxifylline is superior for motility stimulation of cryopreserved sperm.

Maturational Asynchrony between Oocyte Cumulus-Coronal Morphology and Nuclear Maturity in Gonadotropin-Releasing-Hormone Agonist Stimulations

OBJECTIVE To determine oocyte meiotic maturity and asynchrony between cumulus-coronal morphology and nuclear maturity after gonadotropin-releasing hormone agonist (GnRH-a) and norethindrone-programmed stimulations. DESIGN Oocyte meiotic maturity was evaluated at follicular aspiration in 4,961 oocytes after GnRH-a/follicle-stimulating hormone (FSH)/human menopausal gonadotropin stimulations (hMG) for in vitro fertilization patients and 299 oocytes after norethindrone-programmed clomiphene citrate (CC)/hMG in oocyte donors. Maturational asynchrony between the oocytes cumulus-coronal morphology and nuclear maturity was evaluated in 2,336 oocytes. SETTING In vitro fertilization program at the University of Iowa Hospitals and Clinics; academic tertiary care center. INTERVENTIONS After evaluating oocyte cumulus-coronal maturity, cumulus masses were spread to determine oocyte nuclear maturity. RESULTS Fourteen percent, 17%, 50%, 17%, and 2% of oocytes were prophase I, metaphase I, metaphase II, postmature metaphase II, and atretic, respectively. Asynchrony was noted in 28% of prophase I, 71% of metaphase I, 11% of metaphase II, 45% of postmature metaphase II, 32% of atretic, and 28% of all oocytes. Significant differences were not found between GnRH-a and norethindrone-programmed stimulations in asynchrony between cumulus-coronal morphology and nuclear maturity or percentage of prophase I, metaphase I, metaphase II, postmature metaphase II, or atretic oocytes. Sixty-seven percent of oocytes possessed a polar body at retrieval. The rate of fertilization was significantly higher for metaphase II oocytes than postmature metaphase II and metaphase I oocytes > prophase I oocytes. Parthenogenetic activation tended to be highest for postmature metaphase II oocytes. Embryo cleavage was significantly higher for postmature metaphase II, metaphase II, and metaphase I oocytes than for prophase I oocytes. CONCLUSIONS This is the first report of asynchrony between cumulus-coronal morphology and nuclear maturity at follicular aspiration in GnRH-a and norethindrone-programmed stimulations. Asynchrony was observed in 28% of oocytes. A higher percentage of oocytes possessed a polar body at egg retrieval with these stimulation regimens compared with rates reported previously for FSH, FSH/hMG, and CC/hMG stimulations.

Concentration of glycerol required for optimal survival and in vitro fertilizing capacity of frozen sperm is dependent on cryopreservation medium

Sperm survival and in vitro fertilizing capacity were examined following cryopreservation with three concentrations of glycerol (0, 2, and 7.5%) and four cryopreservation media. Post-thaw motility and motility index increased with increasing concentrations of glycerol. Post-thaw velocity, linearity, percentage of zona-free hamster oocytes penetrated, and penetrations per oocyte were greater following freezing with 2 or 7.5% glycerol than with 0% glycerol. A significant interaction between media and glycerol was observed for motility, velocity, linearity, motility index, and percentage of oocytes penetrated. These results suggest that the concentration of glycerol required for optimal survival and in vitro fertilizing capacity of human sperm following cryopreservation is dependent on the type of medium used for freezing.

Conditions of oocyte storage and use of noninseminated as compared with inseminated, nonfertilized oocytes for the hemizona assay

Objectives To examine differences in sperm binding to the zona and recovery of oocytes from the storage vessel after oocyte preservation for the hemizona assay (HZA) by the method currently in predominant use, salt storage at 4°C, as compared with a new method that should allow for indefinite preservation of zona receptors, dimethylsulphoxide (DMSO)/sucrose in liquid nitrogen (−196°C). A second objective was to compare sperm binding to noninseminated zona as opposed to zona from inseminated, nonfertilized oocytes and to examine whether differences in binding potential were related to the patients fertilization rate from the cycle in which the oocytes for the HZA originated. Design Binding and recovery were evaluated after 1, 2, 3, 6, 9, 12, and 17 to 25months of storage. Setting In vitro fertilization and andrology laboratories at the University of Iowa Hospitals and Clinics; academic tertiary care center. Results Binding of sperm was significantly lower for nonfertilized oocytes stored >12months in salt at 4°C than for those stored in liquid nitrogen. Binding was similar after storage for 1, 2, 3, 6, 9, and 12months. Oocyte recovery was significantly lower after storage in salt for >12months as compared with storage in liquid nitrogen. Greater variability in sperm binding was observed between matching zona halves of nonfertilized as compared with noninseminated oocytes. Nonfertilized oocytes also bound fewer total sperm than noninseminated oocytes. The number of sperm bound to noninseminated oocytes was not related to the patients fertilization rate from the cycle in which the oocytes originated. However, significantly fewer sperm bound to the zona of nonfertilized oocytes when the oocyte originated from a cycle in which the patients fertilization rate was >50%. Conclusions These results indicate that storage of oocytes in DMSO/sucrose in liquid nitrogen results in superior long-term (>12months) preservation of zona receptors for sperm binding and improves oocyte recovery as compared with salt storage at 4°C. Although noninseminated oocytes appear to be optimal for use in the HZA, nonfertilized oocytes can be used successfully if the oocytes originate from an IVF cycle in which the fertilization rate is ≤50%.

Treatment of severe male-factor infertility with high concentrations of motile sperm by microinsemination in embryo cryopreservation straws

A microinsemination technique was evaluated for treating our programs most severe cases of male-factor infertility. Oocytes were inseminated with high concentrations of motile sperm (1 to 9×106/ml) in 10 to 150 μl within embryo cryopreservation straws. Fertilization was obtained in 20 of 29 (69%) couples treated by this technique. In the 15 patients in which only embryos generated from the straw technique were transferred, 7 clinical pregnancies resulted (46.7% per transfer). The implantation rate for couples receiving embryos from the straw technique only (12/58; 20.7%) compared favorably to that observed for other cases treated during this same time period with regular insemination techniques (111/766; 14.5%). Clinical pregnancy rates per transfer for IVF-ET, TET, and PROST were 33.0% (1/3), 0% (0/2), and 60.0% (6/10), respectively. The percentage of polyploidic embryos was significantly lower (P<0.0001) for male-factor patients treated by the straw technique with high sperm concentrations that for non-male-factor patients treated during this same time period with standard sperm concentrations. Normal births have resulted from straw inseminations with 3.4×106 and ongoing pregnancies with 5.0×106 motile sperm/ml. The results of this study suggest that some cases of male-factor infertility can be successfully treated by insemination with high concentrations of motile sperm in embryo cryopreservation straws. A technique of centrifuging sperm in straws was also developed to concentrate the entire fraction of washed sperm into 10 μl. Further development of this technique may allow treatment of more severe cases of oligo/asthenospermia by microinsemination with high concentrations of motile sperm than is presently possible with standard washing techniques.

Culture of cytomegalovirus from frozen-thawed semen

Twenty-four donors in a TID program were tested for previous exposure to CMV. Four (16.7%) donors were seropositive for CMV. One donors semen was culture-positive for CMV following cryopreservation and storage at -196 degrees C for up to 9 months. Culture reports for blinded specimens from the same ejaculate were all in agreement. Days to viral detection following inoculation of test cells were similar for specimens from the same ejaculate. Seminal quality was not adversely affected during the period of viral shedding. This appears to be the first report of survival of this previously reported cold labile virus in frozen-thawed semen.

Antibody binding to greater than 50% of sperm at the tail tip does not impair male fertility

A prospective study was conducted with a man displaying 57.5 +/- 13.4% tail tip-directed ASAs to relate results of in vitro tests with attempts to conceive. Tests of seminal quality, penetration of zona-free hamster oocytes, and in vitro penetration of preovulatory cervical mucus were normal. A pregnancy was achieved by natural intercourse during the first cycle in which conception was attempted. More than 50% of sperm were bound by ASAs during the cycle in which conception occurred. These results suggest that ASA binding to certain regions of the sperm surface have no adverse effect on fertility. Therefore, when diagnosing the fertility status of males with ASAs, it appears prudent to use tests that permit determination of the regional distribution of ASA binding to sperm, rather than tests that only permit determination of the presence or absence of ASAs in test fluids.

Survival of Human Sperm Following Controlled- and Noncontrolled-Rate Cryopreservation/Die Überlebensrate menschlicher Spermatozoen nach einer kontrollierten und nicht kontrollierten Kryokonservierung

Summary: The purpose of this study was to develop a noncontrolled‐rate (NCR) method of freezing semen in cryovials which could be used as a backup to controlled‐rate (CR) freezing when the CR freezer was being utilized for freezing of human embryos or oocytes. Semen was frozen with three concetrations of glycerol (0 %, 3.75 %, 7.5 %) in cryoprotective media in a CR freezer or using a newly developed NCR method of freezing. Semen was thawed in 40 °C water or in air at room temperature (RT). Controlled‐rate freezing and 40 °C thawing resulted in significantly greater postthaw sperm motility and motility index compared with NCR freezing and RT thawing. Sperm velocity and linearity were not significantly effected by rate of freezing or thawing except when there was no glycerol present in the cryoprotective media. Postthaw motilities following CR‐40°C, NCR‐40°C, CR‐RT and NCR‐RT freezing‐thawing were 25.6%, 24.8%, 21.6% and 18.3 %, respectively. Because NCR freezing resulted in only a 0.8 % decline in motility compared with CR freezing when semen was thawed at 40 °C it appears that this new NCR method of freezing can be successfully used as a backup to CR freezing when the CR freezer is being used for embryo or oocyte cryopreservation.

Use of oocytes from anonymous, matched, fertile donors for prevention of heritable genetic diseases.

Heritable genetic diseases can be prevented with the use of donor oocytes. We report our experience in using donor oocytes from anonymous, matched, fertile donors in four women with heritable genetic disorders. Our results show that use of donor oocytes is a practical, successful, and currently available technique for the prevention of genetic disorders.