Lars Hamberger

Infertility therapy associated-multiple pregnancies (births): an ongoing epidemic

Multiple gestation is now recognized as a major problem associated with both assisted reproductive technologies (ART) and also with ovulation induction therapies. Although some countries are beginning to adopt measures to address this issue, either through legislation or the development of clinical guidelines, there is a clear need to ensure recognition and a consistent approach to this problem worldwide. In particular, there is a need to educate both healthcare professionals and the lay population that multiple gestations are not a desirable outcome for the infertile couple.

Collection of human oocytes by the use of sonography

A technique for ultrasonically guided percutaneous oocyte aspiration was developed utilizing standard real-time, linear-array ultrasound equipment. Forty-four patients attending our in vitro fertilization and embryo transfer (IVF-ET) program were included in this study. In 38 patients, follicular puncture was performed under general anesthesia and in 6 cases performed under local anesthesia. Fifty-two follicles with a mean diameter greater than or equal to 18 mm were punctured, and 40 mature oocytes were recovered corresponding to a success rate of 77% per follicle. Six of the punctured follicles were considered cystic when the aspirated granulosa cells were examined microscopically and, if these were excluded, the corrected recovery rate was 87% per follicle. In two patients, ovulation had occurred at the time for oocyte collection. In one of these patients, a mature oocyte was, however, recovered from the pouch of Douglas by the use of sonography. On the basis of these results, ultrasonically guided puncture of follicles for collection of human oocytes in our IVF-ET program seems suitable for use in all cases where laparoscopy is presently used and, moreover, in cases with severe adhesions, where laparoscopy may fail.

Transvesical and Transvaginal Approaches for the Aspiration of Follicles by Use of Ultrasound

The use of ultrasound to study follicular growth in both normal and stimulated cycles is well established.’-’ The technique is today probably utilized in all in vitro fertilization and embryo replacement (IVF/ER) programs as a complement to hormone analyses for prediction of the optimal time of oocyte c~l lec t ion .~’ Oocytes have, hitherto, mainly been recovered by means of laparo~copy.*-’~ However, radiologists and ultrasonographers have used ultrasound for many years to guide the needle for percutaneous puncture of intra-abdominal structures.” One of the advantages of using this technique is that it is less invasive than other methods if the only purpose is to empty, as with cysts, or to retrieve cells for cytological evaluation, for example. Analogous with this principle, the preovulatory follicle is a cystic structure and the goal in this case is to retrieve intrafollicular cell material for culture purposes. Thus, ultrasound in this connection seems to be a self-evident alternative to use of laparoscopy. Although human IVF/ER has been established clinically since the end of the 1970s for treatment of certain types of female and male infertility, ultrasound-guided follicle aspiration has not been utilized for more than 3 years.’6s” However, the vast increase in experience with this method during the last 2 years indicates that the technique is here to stay as an alternative or complement to laparoscopy.’” This paper will deal with the ultrasound-guided method for oocyte retrieval as it has been developed and utilized in the IVF/ER programs in Sweden. Future possible developments within the technique will also be discussed.

Fertilization and pregnancy after intracytoplasmic microinjection of acrosomeless spermatozoa

Spermatozoa lacking acrosomes were injected into the cytoplasm of mature human oocytes. In two subsequent cycles, 12 of 28 (43%) oocytes were fertilized, and the ET of the second cycle resulted in a twin pregnancy. This report describes, to the best of our knowledge, the first case of successful fertilization and delivery after using acrosomeless spermatozoa. Our hope is that with increasing experience with microinjection, in the near future, this type of infertility will not remain a serious problem.

Effects of a low-dose desogestrel-ethinylestradiol combination on hirsutism, androgens and sex hormone binding globulin in women with a polycystic ovary syndrome.

Abstract. Twenty women suffering from a polycystic ovary syndrome (PCO) accompanied by hirsutism were given a low‐dose oral contraceptive combination containing 0.150 mg desogestrel plus 0.030 mg ethinylestradiol for 8 months. The pretreatment situation regarding hair and hormone profiles in the PCO group was compared with that in 22 regularly menstruating women. Serum levels of free and total testosterone and androstenedione were significantly elevated in PCO women, as were body weight, blood pressure, hair diameter and depilation frequency. Sex hormone binding globulin (SHBG) binding capacity was lower. Following treatment of the PCO group for 8 months, total and free testosterone levels were depressed, but androstenedione had not changed significantly. SHBG binding capacity was increased five‐fold. Body weight decreased in the obese women. Hair growth was significantly suppressed and the hair itself was less coarse. Depilation intervals were longer. Acne, present before the treatment had now disappeared. Blood pressure did not change. Few and mild side effects were recorded. After treatment, 3 women succeeded in becoming pregnant and in 8 others spontaneous menstruations had recurred.

Contractile Patterns of Isolated Oviductal Smooth Muscle Under Different Hormonal Conditions

The isthmic portion of the human fallopian tube was excised during surgery and the ampullary-isthmic junction was identified. Muscle strips (1-mm wide) were dissected from the outer longitudinal and the inner circular layers at this region and used for isometric recordings of spontaneous contractile activity in organ bath experiments. It was found that the amplitude and the duration of individual contractions were similar during various phases of normal menstrual cycles, whereas the frequency of contractions was significantly increased during the periovulatory period in both circular and longitudinal muscle. During early pregnancy or treatment with combined oral contraceptives the motility pattern was similar to that observed in the late luteal phase, being characterized by a comparatively low contraction frequency. In the perimenopausal period, irregular patterns were frequently observed whereas preparations obtained from postmenopausal women exhibited a very weak activity with low amplitude and frequency of contractions. Similar activity was encountered in specimens from patients treated with androgens. It is concluded that endogenous estrogens stimulate human oviductal contractility while endogenous progesterone has a depressive action on tubal activity. Both effects occur with a certain delay and are prolonged for up to 3 days, i.e., the changes in the serum levels of ovarian steroids and the induced alterations of tubal contractility are out of phase.

Ultrasound studies of vascular and morphological changes in the human uterus after a positive self-test for the urinary luteinizing hormone surge

The aim of the study reported here was to establish complementary data for changes in uterine size, echogenicity and vascularity during the menstrual cycle relative to a positive self-test for urinary luteinizing hormone (LH) and day 1 of next menses. Thirteen volunteers (aged 23-32 years) with apparently regular menstrual cycles were recruited from the nursing staff. The plan was to examine all women by transvaginal ultrasonography with colour Doppler imaging on day 11 of the menstrual cycle. A urinary LH self-test was to be used daily until a positive result was obtained and the women were to be re-scanned daily until the dominant follicle had ruptured. All women were then to be scanned at least every 48 h (within +/- 2 h of the same time of day) until day 6 of the next menstrual cycle. Matched samples of peripheral blood were taken at the time of each scan for hormone analysis. The main outcome measures were the times of follicular rupture, a positive test result for urinary LH and the start of menses, uterine volume, cavity length, endometrial thickness and grade, pulsatility index (PI), and time-averaged and peak systolic maximum velocities in uterine and radial arteries and in subendometrial vessels. Nine women fulfilled the criteria for an ovulatory cycle, and seven provided data over the complete study. The principal changes relative to a positive urinary LH test were (i) a continued rise in endometrial thickness to days 3 and 4 (this index then remained relatively constant, but the layered appearance was lost) and (ii) a gradual decrease in the uterine arterial PI. There was a significant rise in uterine volume, cavity length and uterine arterial PI around the time of the next menses, and a fall in endometrial thickness and blood velocity in the uterine and radial arteries and subendometrial vessels. The data may have implications for the assessment of reproductive status and the design of future studies on disorders of implantation or menstruation.

IS PATERNAL MITOCHONDRIAL DNA TRANSFERRED TO THE OFFSPRING FOLLOWING INTRACYTOPLASMIC SPERM INJECTION

During intracytoplasmic sperm injection (ICSI) the whole sperm, including head, midpiece and tail, is injected into the middle area of the oocyte. To find out what happens to the sperm mitochondria after ICSI, we checked the first six children born after ICSI treatment for occurrence of paternal mitochondrial DNA (mtDNA). The difference between maternal and paternal mtDNA in the investigated couples in our study was confined to single-base pair substitutions and we had to rely on restriction enzyme cleavage to differentiate between the mitochondrial genomes of the parents. With this kind of assay we were able to reach a sensitivity of about 0.2% for the paternal mtDNA. However, as uneven partition between tissues of heteroplasmic mtDNA is expected to occur, it would not be unlikely that an enrichment to 0.2% would occur in a given tissue if paternal mtDNA was transmitted by the ICSI procedure. We did not detect this level in the blood in any of the six children.

Reinsemination of one-day-old oocytes by use of intracytoplasmic sperm injection *

OBJECTIVE To evaluate the possible advantages of reinseminating oocytes by use of intracytoplasmic sperm injection (ICSI). DESIGN Clinical study. SETTING In vitro fertilization unit with research facilities. PATIENTS Fifty-seven couples who were part of our regular IVF program. INTERVENTIONS Nonfertilized oocytes from IVF cycles with no or very low fertilization were microinjected with spermatozoa approximately 25 hours after oocyte pick-up. MAIN OUTCOME MEASURES Fertilization and pregnancy rates. RESULTS A mean fertilization rate of 46.5% was obtained when reinseminating the oocytes on day 2 using the ICSI procedure. Of 57 cycles with completely or almost completely failed fertilization, 29 patients received ET after reinsemination by ICSI. Two of these transfers resulted in pregnancies (6.9% per ET) and two healthy babies were born. CONCLUSION Despite this relative success, considering both the extra work involved and the potential genetic risk, it is doubtful whether ICSI on day 2 should be recommended as a routine procedure. For training and research purposes, however, this approach can be of value.

Human blastocysts for the development of embryonic stem cells

Establishment of human embryonic stem cells (hES) from surplus human IVF embryos has been successful when both fresh and frozen-thawed cleavage stage embryos have been cultured to the blastocyst stage. This study reports the characteristics of the starting material, the blastocysts, for hES cell lines that were first derived at the University of Gothenburg, Sahlgrenska University Hospital in 1999. Twenty-two hES cell lines were derived by Cellartis AB from 114 blastocysts, giving an overall success rate of 19.3%. The blastocysts from which the hES cell lines were established were of varying morphological quality, both fresh and frozen-thawed. Two techniques of hES establishment were applied, i.e. direct application of the blastocysts on feeder cells or the standard immunosurgery method. It was further found that the efficiency by which frozen-thawed embryos gave rise to new hES cell lines was 3.7 times better than with fresh surplus embryos. These findings suggest that frozen-thawed embryos are superior to fresh surplus human embryos in hES cell establishment, which also avoids specific ethical problems associated with embryo donation in a fresh IVF cycle.