Effect of insulin-like growth factors (IGFs) and IGF-binding proteins on in vitro sperm motility.

OBJECTIVE: Previous studies have shown that exogenous growth hormone (GH) produces increases in sperm motility when given to subfertile men. Previous studies have also demonstrated the presence of IGFs and IGFBPs in seminal plasma. We have therefore investigated the effects of insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein 2 (IGFBP-2) and intact IGFBP-3 on in vitro sperm motility. DESIGN AND METHODS: Using computer-aided sperm analysis, we investigated the effects of IGFs and IGFBPs on the in vitro sperm motility parameters: curvilinear velocity (CV), progressive velocity (PV), linearity (Ln), straightness (St), amplitude of lateral head movement (ALH), and beat frequency (BF). Washed motile sperm selected by the 'swim-up' method, from normozoospermic samples, were incubated at 37 degrees C in 5% CO2 in air with IGF-I, IGF-II, IGFBP-2, IGFBP-3, or control Earle's media, and were examined at time 0 and after 60 min incubation. Changes in motility parameters after 60 min incubation were compared with controls by analysis of variance (ANOVA). RESULTS: Compared to controls, statistically significant changes occurred at time 60 min after incubation: IGF-I decreased CV and ALH significantly (P < 0.05), but IGFBP-3 increased Ln, St, BF, and decreased ALH significantly (P < 0.05). In contrast, IGF-II, IGFBP-2, and a combination of IGF-I/IGFBP-3, had no significant effects. CONCLUSIONS: IGF-I and IGFBP-3 have differing and opposing effects on in vitro sperm motility parameters and thus may have a role in modulating in vivo sperm motility.

The first frozen embryo donation pregnancy for hypergonadotrophic hypogonadism in Singapore--hormonal profile and obstetric outcome.

This is the first report in South East Asia of a singleton frozen embryo donation pregnancy for hypergonadotrophic hypogonadism. The hormonal profile was compared with that of a control group of normal uncomplicated singleton pregnancies in Singapore. The plasma beta hCG levels were lower compared to those of our normal uncomplicated singleton pregnancies at 2 to 3 weeks after the embryo transfer but became comparable at 4 to 5 weeks after embryo transfer. The successful vaginal delivery and the obstetric complications developed in this case are discussed.

Stimulation regimens in Assisted Reproductive Technology (ART) programme: experience in the University Hospital, Singapore.

Ovarian stimulation is critical to the success of patients in Assisted Reproductive Technology (ART) programmes. We compared two stimulation regimes retrospectively for ART in the NUH programme. These were the 2:1 and the GnRHa-FSH/hMG regimes. The former was our first-line regime while the latter was used for cycles where there was a prior endogenous LH surge, previous poor response, or an elevated LH level between days three to five of the cycle. All cycles in our ART programme in 1991 were studied, except those for special research procedures, e.g. micro-insemination sperm transfer (MIST) cycles, a total of 241 cycles. Cancellation rates were 14.4% (21 of 146 cycles) and 37.3% (19 of 51 cycles) for the 2:1 and GnRHa-FSH/hMG regimes respectively (p > 0.001). For the 2:1 regime, the majority of cancellations were due to ovulation prior to the oocyte recovery (42.9%; nine of 21 cycles). However, for the GnRHa-FSH/hMG regime, almost all the cancellations were due to poor response (84.2%; 16 of 19 cycles). Fertilisation rates were lower for the 2:1 regime (for both IVF-ER and IVF-TET, where sperm quality was poorer) compared to the GnRHa-FSH/hMG (55.0 and 66.6% respectively; p < 0.001). Pregnancy rates were higher for the 2:1 regime when IVF-ER and IVF-TET were used (16.4% and 23.3% respectively per oocyte recovery, versus 9.8% and 18.8% respectively for the GnRHa-FSH/hMG regime).

Micro-insemination sperm transfer (MIST) and its application to male subfertility: current strategies to improve results.

It can be difficult to achieve a pregnancy for patients with severe male factor subfertility. Hence, micro-manipulation techniques, have been applied to this problem. Direct deposition of sperm into the oocyte under the zona (micro-insemination sperm transfer, MIST) has improved the fertilisation rates, but pregnancy rates have been very low. This paper describes the possible new strategies to improve the technique currently. They include methods to improve sperm recovery, to improve acrosome reaction, to improve the quality of embryos by using co-cultures and to wait for good cleavage before transfer, and to improve luteal phase support. There are also many new techniques being developed which may contribute to further pregnancy successes. They include the use of electro-fusion, laser-fusion and the Xenopus cell-free system.

Human ampullary co-cultures for blastocyst transfer in assisted reproduction.

Although the assisted reproductive techniques (ART) have contributed significantly over the last decade in alleviating subfertility in the childless couple, the implantation and take-home baby rates have been stubbornly low. A major cause for such low success rates has been the reduced viability of replaced embryos perhaps induced by the suboptimal in vitro conditions used in ART laboratories. One approach to improving embryo viability is to provide the growing embryo with a simulated in vivo environment by replicating the conditions existing in the human fallopian tube in vitro. This requires either the maintenance of an intact fallopian tube in vitro or establishment and maintenance of tubal epithelial cell-lines which could be used as feeder layers for early embryonic growth. The concomitant growth of cells with embryos in vitro has been referred to as co-culture. This paper discusses the in vitro behaviour of human tubal epithelial cells, the fertilisation and growth of embryos in ampullary co-culture, the specificity of co-cultures, the mechanism of action of co-cultures and the methods of screening the human ampullary co-culture system for microbes. The pregnancy and implantation results on 50 patients enrolled for a co-culture clinical trial are presented and the future use of this system discussed.

Future of assisted reproductive technologies.

Within a decade, from the birth of Louise Brown in 1978, there has been tremendous advances in the field of assisted reproduction. These advances are the collective result of techniques known as Assisted Reproductive Technologies (ART). ART is defined as techniques in which the oocyte is handled in-vitro before replacement either as oocytes or embryos. The techniques in ART include in-vitro fertilisation and embryo replacement (IVF-ER), gamete intra-fallopian transfer (GIFT), tubal embryo (TET), donor oocytes and embryos, and freezing of embryos and oocytes. The most recent has been assisting fertilisation by micro-manipulation, including zonal procedures and micro-insemination, e.g. micro-insemination sperm transfer (MIST). There has also been many ethical issues in ART, and little advance in the "take home" baby rate. There is now major interest in co-culture of embryos with ampullary, endometrial and fibroblast mono-layers, to improve the quality of these embryos before their return into the mother. Because of the public's interest in ART, its usefulness has been "oversold". In time to come, ART may be limited to patients who really need it.

Subzonal transfer of multiple sperm (MIST) into early human embryos.

Microinsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6-24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome-intact and acrosome-reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm-blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane-bound vesicles. Both acrosome-intact and acrosome-reacted sperm heads were found in vacuoles. Acrosome-reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.

Micro-centrifugation of human spermatozoa: its effect on fertilization of hamster oocytes after micro-insemination spermatozoal transfer.

Micro-centrifugation (MC) at 6500 r.p.m. (3352 g) has not been used previously for spermatozoal concentration and subsequent fertilization. We investigated MC for micro-insemination spermatozoal transfer (MIST) of human spermatozoa from normal donors into hamster oocytes. MC resulted in reduced penetration of hamster oocytes, both after MIST [77.4% (41/53) versus 87.8% (43/49) for control; NS] and after exposure to zona-free hamster oocytes [60.8% (79/130) versus 72.7% (88/121) for control; P less than 0.05]. However, MIST under the zona resulted in better incorporation of sperm nuclei when compared with zona-free hamster oocytes, for spermatozoa exposed to micro-centrifugation (77.4 versus 60.8%, P less than 0.05) and controls (87.8 versus 72.7%, P less than 0.05). Polyspermy was higher after MIST [22.0% (9/41) versus 13.9% (11/79); NS] for MC+, and [25.6% (11/43) versus 13.6% (12/88); NS] for MC-. We conclude that MC does have a negative, but minimal effect on the fertility of human spermatozoa with respect to hamster oocytes.

The Sertoli cell of the water buffalo--an electron microscopic study.

The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity.

Micro-insemination: genetic aspects.

Micro-insemination involves sperm deposition directly into oocytes. This can be by transfer of sperm (Micro-Insemination Sperm Transfer, or MIST) or by micro-injection into the ooplasm (Micro-Insemination Micro-Injection into Cytoplasm, or MIMIC). Micro-insemination is indicated in spermatozoa with no or very poor motility, very low density, multiple defects, or inability to penetrate oocyte vestments. There is a 10% incidence of chromosomal abnormalities in spermatozoa from fertile and normal men. However, there is no increase in sperm chromosomal abnormalities in men with normal peripheral karyotypes and highly abnormal sperm parameters. Preliminary results of karyotypes of human oocytes that failed to become fertilized after MIST and mouse morulae and blastocysts produced after MIST reveal that there was no significant increase in aneuploidy or polyploidy. There is evidence that MIMIC may result in increased abnormal sperm karyotypes. Polyspermy is low in the mouse and human after transfer of multiple spermatozoa into the perivitelline space, thus suggesting an oolemmal block. However, blastomere membranes do not fuse with spermatozoa, as observed in a study of MIST into human embryos. Zona drilling with acid is not advised because of disturbances to chromosomal kinetics. The conclusion of this review is that MIST does not result in an increased risk of chromosomal abnormalities, while caution must be exercised with MIMIC.

Identification of crossbred buffalo genotypes and their chromosome segregation patterns.

Chromosome analysis on different breed types of water buffaloes (Bubalus bubalis) was undertaken to identify their karyotypes and to determine the pattern of chromosome segregation in crossbred water buffaloes. Altogether, 75 purebred and 198 crossbred buffaloes including 118 from Malaysia and 80 from the Philippines, were analyzed in this study. The diploid chromosome number of the swamp buffalo from both countries was 48 and that of the river buffalo was 50, while all F1 hybrids exhibited 49 chromosomes. The F2 hybrids consisted of three different karyotype categories (2n = 48, 2n = 49, and 2n = 50), whereas the backcrosses included two different karyotype categories each, with 2n = 48 and 2n = 49 in the three quarters swamp types and 2n = 49 and 2n = 50 in the three quarters river types. Chi-square tests on pooled data from Malaysia and the Philippines indicated that the distribution of different karyotype categories of F2 animals did not deviate significantly from the 1:2:1 ratio expected if only balanced gametes with 24 and 25 chromosomes were produced by the F1 hybrids. In the three quarters swamp and three quarters river types, the respective karyotypic categories were in ratios approximating 1:1. The distribution of chromosome categories among the F2 hybrids and backcrosses suggests that only genetically balanced gametes of the F1 hybrids are capable of producing viable F2 and backcross generations.

Birth from replacement of frozen-thawed embryos after failure of gamete intra-fallopian transfer: a successful pregnancy case.

The patient was 8 years subfertile and had failed other forms of treatment when she was enrolled in the GIFT program. Of the total of 16 oocytes recovered 4 were transferred and the remaining 12 inseminated with her husband's sperm. Four resulting embryos were frozen. When she did not conceive, the 4 embryos were thawed 3 months later and replaced into her. She conceived and recently delivered a pair of twins. The protocol will be discussed in detail. Cryopreservation of embryos therefore may increase the patient's chance of pregnancy in a GIFT program.

Human fertilization by micro-injection of immotile spermatozoa.

Microfertilization of human oocytes with spermatozoa from a man with immotile cilia syndrome is reported, confirming a preliminary investigation where a zona-free donor oocyte was fertilized with spermatozoa from the same patient. Oocytes from his spouse were obtained by laparoscopy after routine stimulation with clomiphene citrate, human menopausal and chorionic gonadotrophins, and were cultured for 4-6 h in Whittingham's T6 medium, supplemented with 10% of her serum. The spermatozoa were washed and processed in the same medium and capacitated for 6-8 h before micromanipulation. Three of five mature oocytes were fertilized by micro-injection of a single immotile spermatozoon into the perivitelline space. One oocyte produced a two-pronuclear ovum assessed 19 h after injection, while the other two produced 2-cell embryos with blastomeres of equal size, 22 h after injection. These embryos cleaved to 3-8-cell stages in culture before embryo replacement. No pregnancy resulted from embryo transfer. The results conclusively demonstrate that human oocytes can be fertilized successfully with immotile spermatozoa by micro-injection and the work has profound implications in the treatment of severe male infertility.

Effect of sperm motility on human in vitro fertilization.

Several sperm motility parameters in semen prepared by the swim-up technique were compared with IVF rates in 84 patients. The patients were either on clomiphene + human menopausal gonadotrophin or follicle stimulating hormone + human menopausal gonadotrophin stimulation regimens. Motility ratings were assessed both manually according to World Health Organization guidelines as well as computer-automated semen analysis (Cellsoft, Cryoresources, USA). Motility ratings of greater than or equal to 2 yielded significantly higher fertilization rates (78-82%) than ratings below 2 (20-23%) (p less than 0.001) for patients on both regimens. Velocity (41, 55, 78 microns/sec) and mean amplitude of lateral head displacement (1.96, 3.29, 4.91 microns) correlated significantly with and between manual ratings of 1, 2, and 3, respectively (r = 0.83; p less than 0.01). No significant differences were observed in linearity and beat/cross frequency between the manual ratings, although beat/cross frequencies tended to reduce linearly with increases in intensity of motility. The velocity of sperm motility has a significant effect on fertilization rates, and cut-off points of greater than or equal to 2 or greater than or equal to 50 microns/sec predict the actual potential and likely success of in vitro fertilization. These criteria on the swim-up semen should be used in the selection of patients admitted to IVF programs, and they justify the necessity of research investigations to improve motility in those patients with sluggish motility.

Chromosomes of gaur cross domestic cattle hybrids.

The chromosomes of five gaur (Bos gaurus hubbacki) domestic cattle (B indicus cross B taurus) hybrids (three females, two males) were studied using the leucocyte culture method and centromeric (C) banding technique. All the hybrids had a diploid chromosome number of 2n = 58, made up of two submetacentric autosomes (different in size) and 54 acrocentric autosomes, most of which could be arranged in pairs in descending order of size. The sex (X) chromosomes in females were a pair of submetacentric chromosomes smaller than the submetacentric autosomes. The Y chromosome in males was a small submetacentric chromosome. The C banding patterns were useful in identifying the X and Y chromosomes and the inherited submetacentric autosomes from the gaur sire. Phenotypically, the hybrids resembled normal B indicus cross B taurus calves except for the presence of a distinct hump-like dorsal ridge containing the spinous processes of the third to 11th thoracic vertebrae, upright 'deer-like' ears and long lean legs. The potential of these hybrids as important genetic resources for meat production is stressed.

Relative importance of larval and adult Mecistocirrus digitatus in inducing resistance to a reinfection in calves.

Calves immunized with adult Mecistocirrus digitatus implanted directly into the abomasum did not develop a substantial degree of immunity to a subsequent large oral (challenge) dose of larvae, which developed to maturity. In contrast animals immunized by oral infection developed strong resistance. The calves implanted with adult worms appeared to show a greater degree of susceptibility to maturation of the challenge infection than controls which received a challenge of the same magnitude without any previous immunization. The implanted female adult worms established in the hosts and continued to produce more eggs for a longer time than those which developed to maturity from the oral immunizing infection with third-stage larvae. Passive haemagglutination studies revealed that the implanted adult worms stimulated little or no antibody response in the hosts. In the calves which did not show a response to the adult worm implant the subsequent challenge with an oral infective dose of third-stage larvae also failed to stimulate a response. Likewise the two calves from the group which showed a weak antibody response to the adult worm implant did not show an increased response when challenged. In contrast, calves immunized with an oral infection of third-stage larvae had an antibody response which showed a vigorous rise on challenge in four of the five calves. Thus a direct relationship between resistance to challenge infection and the antibody response determined by the passive haemagglutination and gel-diffusion tests was observed in the calves immunized orally.