Bruce A. Sandow

Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization.

Oocytes of varying stages of maturity were aspirated from follicles primed with either human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) or a combination of follicle-stimulating hormone (FSH), hMG and hCG. Of the aspirated oocytes from 44 cycles, 74 were considered to be immature by virtue of morphologic characteristics of the oocytes and the degree of intercellular expansion of the associated cumular and membrana granulosa cells. After incubation periods of 22 to 35 hours in a Hams F-10-based culture medium, these immature oocytes were inseminated with sperm donated by the patients husband. Ultimately, 44 conceptuses were transferred to the respective uteri of 30 patients. Eight pregnancies were established as a result of these 30 transfers, two of which resulted from the transfer of only developed immature oocytes.

The program for in vitro fertilization at Norfolk

Several aspects of the program of in vitro fertilization (IVF), or, as it is called in Norfolk, the program for the Vital Initiation of Pregnancy (VIP), have been or are in the process of publication. However, because there has been no overall account, it seems appropriate to give a brief report of a general nature covering the period from the beginning of the effort in late February 1980 through December 31, 1981. Although minor changes were constantly made in the protocol, there were two major revisions. Therefore, a discussion of the program during three distinct periods, i.e., 1980, 1981—Phase I, and 1981—Phase II, is necessary. During 1980 and 1981 all patients had either no fallopian tubes or irreparable tubes.

Three years of in vitro fertilization at Norfolk

During the 3 years from 1981 to 1983, 319 consecutive patients in 560 cycles were treated in a program of in vitro fertilization at Norfolk. All patients were stimulated by human menopausal gonadotropin supplemented by human chorionic gonadotropin. There were transfers in 429 cycles, resulting in 105 pregnancies. Over the 3-year span, the pregnancy rate by cycle was 19%; by transfer, 25%; and by patient, 33%.

The importance of the follicular phase to success and failure in in vitro fertilization

One hundred seventy-five cycles in patients with irreparable tubal disease were stimulated by human menopausal gonadotropin/human chorionic gonadotropin for the purpose of in vitro fertilization. As judged by the height of the peripheral estradiol response, the patients were classified as high, intermediate, or low responders. In addition, the estradiol pattern of the response was found to be separable into six categories. The pregnancy rate was found to be related to the height and to the pattern of peripheral response. The overall pregnancy rate in this consecutive series was 19% but varied according to the height and pattern of response from 40% to 0%.

Preclinical models for human pre-embryo biopsy and genetic diagnosis. I. Efficiency and normalcy of mouse pre-embryo development after different biopsy techniques

OBJECTIVE To compare the usefulness of three micromanipulative methods at two different stages of pre-embryo development and to assess possible effects on postbiopsy survival and development. DESIGN Four-cell and eight-cell mouse pre-embryos were biopsied using enucleation, aspiration, or extrusion of single blastomeres. After biopsy, pre-embryos were observed for in vitro and in vivo development. SETTING Laboratories of The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. PATIENTS, PARTICIPANTS Only mice were used. INTERVENTIONS Pre-embryo biopsy, developmental normalcy and pre-embryo transfer were studied. MAIN OUTCOME MEASURE(S) Few pre-embryos died as a result of biopsy trauma. High postbiopsy survival rates were associated with normal intrauterine and postnatal development. RESULTS Expanded blastocyst formation rates from four-cell and eight-cell pre-embryos were 94.6%, 96.7% (controls); 80.7%, 89.1% (enucleation); 90.1%, 91.7% (aspiration); 83.1%, 91.5% (extrusion), respectively. Live birth rates at the four-cell stage were slightly lower in the enucleation group than in the blastomere aspiration and extrusion groups or controls (49.2% versus 58.8%, 56.3% and 66.7%, respectively). For the eight-cell stage, there were no differences between the groups. No developmental abnormalities were found in body or organ weights, in neonates or at 3 weeks of age, or in their subsequent ability to reproduce a second generation. CONCLUSIONS Biopsy of mouse pre-embryos produces only a small loss of viability because of trauma and permits normal prenatal and postnatal development among surviving pre-embryos.

Vital initiation of pregnancy (VIP) using human menopausal gonadotropin and human chorionic gonadotropin ovulation induction: Phase II—1981

Laparoscopies for oocyte aspiration in 31 cycles were performed on 25 patients receiving human menopausal gonadotropin and human chorionic gonadotropin. Sixty oocytes were aspirated, of which 48 were considered preovulatory. Ninety-seven percent (58 of 60) of the oocytes were found in the original aspirate, and the remaining oocytes were found in either the first or second follicle wash. The fertilization rate per preovulatory oocyte was 33% (16 of 48), whereas on a per cycle basis it was 39% (12 of 31). A total of 15 conceptuses (2-cell = 5; 3-cell = 3; 4-cell = 7) were transferred to 12 patients, and two pregnancies were established. These pregnancies were established by transfers of 3-cell and 4-cell conceptuses at approximately 47 hours after insemination. Both pregnancies resulted in term deliveries of normal infants.

Correlation of human menopausal gonadotropin/human chorionic gonadotropin stimulation and oocyte quality in an in vitro fertilization program*

One hundred forty-seven cycles in normal ovulatory patients are reported. All were stimulated with human menopausal/human chorionic gonadotropin. Three estrogen responses were identified: normal, high, and low. Patients who achieved pregnancy formed a fourth category, the pregnancy group. The number of preovulatory and immature oocytes, the preovulatory and immature oocytes that fertilized normally or abnormally, the ones that cleaved in culture, and the ones that were transferred were used as parameters to compare quality of the oocytes in each of the estrogen responders. No significant differences were found in any of them. Abnormal zonae pellucidae are described as possibly due to overmaturation of the follicle. No significant difference in the proportion of abnormal zonae in the different categories was found.

Enhancement of the developmental potential of mouse oocytes matured in vitro by gonadotropins and ethylenediaminetetraacetic acid (EDTA)

We attempted to improve the developmental potential of mouse oocytes matured in vitro. First, the effect of gonadotropin supplementation of the oocyte maturation medium was tested. The addition of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) alone significantly increased the rate of development of inseminated oocytes to two-cell embryos, resulting in a twofold increase in blastocyst development. There was no significant difference between FSH and LH supplementation. However, the beneficial effect of FSH or LH was abolished when both were added together. Next, we tested the effect of ethylenediaminetetraacetic acid (EDTA) supplementation of the embryo culture medium. The addition of 10 μM EDTA significantly enhanced the development of embryos derived from oocytes matured in vitro, both to two-cell embryos and to blastocysts. These data suggest that the inadequate development of embryos from oocytes matured in vitro results from a defect similar to that inherent in outbred mouse embryos showing the two-cell block in vitro.

In vitro fertilization in Norfolk, Virginia, 1980-1983

Three years of progress of the Vital Initiation of Pregnancy (VIP) Program in Norfolk is reported. No conception resulted from 41 oocyte aspirations during spontaneous menstrual cycles in 1980. An average of 3.7 oocytes per cycle, or a 73.5% recovery rate, resulted in 362 human menopausal gonadotropin/human chorionic gonadotropin-induced cycles from January 1981 to March 1983. Forty pecent of the oocytes recovered from these cycles were preovulatory, 35% atretic, and 25% immature. Immature oocytes were often matured in vitro, fertilized, and found to produce pregnancies. A total of 62 pregnancies occurred, which represents a 17 or 23% pregnancy rate, based on laporoscopies or embryo transfers, respectively. There were 11 preclinical and 7 clinical miscarriages. Twenty-nine normal babies have been delivered, including a set of twins. The remainder appears to be normally progressing pregnancies. Polyspermia was observed in 8.8% of the fertilizable oocytes.

Full physiological maturation in vitro of immature mouse oocytes induced by sequential treatment with follicle-stimulating hormone and luteinizing hormone.

Cumulus cell-enclosed immature mouse oocytes were matured in medium supplemented with various combinations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol. FSH or LH alone stimulated oocyte maturation, resulting in a significant increase in the rate of development to blastocysts following fertilization in vitro and embryo culture. There was no significant difference between FSH and LH. The effect of FSH was neutralized by FSH antiserum, while that of LH was not indicating that the stimulation of maturation by LH was not due to FSH contamination in the LH preparation. When LH was added after 2 hr of culture with FSH (sequential combination), blastocyst development was significantly increased compared with FSH alone, reaching the same level as the in vivo matured oocytes. The addition of estradiol, 0.1 ng/ml to the sequential combination of FSH and LH had no effect, while 0.01 and 1 ng/ml produced a negative effect. The birth rate of normal live offspring following embryo transfer showed no significant difference between embryos derived from oocytes matured in vivo and in vitro (sequential combination with or without 0.1 ng/ml estradiol) or between the two in vitro treatment groups.