S. S. Ratnam

The sperm centriole: its inheritance, replication and perpetuation in early human embryos

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident. During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blastocysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells.

Cocultures : a new lead in embryo quality improvement for assisted reproduction

One of the contributory causes to poor PRs in assisted reproduction has been the decreased viability of transferred embryos and the transfer of four-cell embryos into an environment that naturally would be receptive only to 5-day-old blastocysts. In this paper, we have reviewed our own work and that of others on the role of tubal ampullary cells (cocultures) to mimic the in vivo environment to bring about improved embryo quality and an increased number of blastocysts for replacement in IVF patients. The establishment, maintenance, and behavior of human tubal cell lines is first presented, followed by their use as cocultures for fertilization and cleavage of embryos. The mode of action, specificity, and cryopreservation of ampullary cells are also discussed. The currently available results of pregnancies after cocultures are presented together with future aspects of research that are necessary to refine the coculture system. The ultimate aim is to mimic in vivo conditions in vitro, so that at least the PRs of assisted conception can be parallel to normal fecundity in the human. Therefore, a very attractive future includes the freezing of blastocysts generated from coculture, thawing, and replacing them in natural cycles.

Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture

OBJECTIVE To evaluate the embryonic behavior in vitro and the pregnancy and implantation rates of embryos grown in a human ampullary cell coculture system. DESIGN In a prospective study, two pronuclei embryos were cultured on human ampullary feeder layers up to the two to six-cell and blastocyst stages and replaced either as tubal, uterine, or sequential transfers. SETTING Assisted reproductive technology program in a university-based hospital. PATIENTS Fifty women with a mean age of 35.6 years who went through a single coculture cycle. Thirty of the patients were admitted for in vitro fertilization (IVF) and 20 for tubal embryo transfer (TET). RESULTS The overall clinical pregnancy rate (PR) for all 50 patients was 44% per cycle (IVF, 37%; TET, 55%) and the implantation rate was 31.8% (IVF, 31.0%; TET, 32.6%). Sixty-eight percent of pregnant patients were over 35 years, and 68% had two previously failed assisted reproduction cycles. Five of 9 patients who received sequential transfers became pregnant. Three of the 22 pregnancies aborted (2 after sequential transfer), and there was one ectopic. Overall, 88% of two to six-cell stage embryos were of good quality. CONCLUSIONS The human ampullary coculture system produces better quality embryos, increased numbers of blastocysts with improved PRs and implantation rates. The beneficial effects of the feeder layer may be through the release of embryotrophic factors and detoxification of the medium by the cells. Coculture is a new concept in assisted reproduction and has tremendous potential in boosting conception rates by mimicking the in vivo environment.

Influence of serum paraoxonase polymorphism on serum lipids and apolipoproteins.

One hundred and sixty‐three healthy Chinese subjects of both sexes were studied for serum paraoxonase (PON) polymorphism, and levels of lipids and apolipoproteins in order to examine effects of PON alleles on these parameters. The level of serum triglyceride was significantly higher in high activity allele (PON*B) compared with that in low activity allele (PON*A) in both sexes (P<0.01). The subjects with PON A had significantly higher LDL cholesterol (P<0.05) and lower Apo A‐II and ApoB levels. The influence of serum paraoxonase on serum lipids was estimated further by Spearmans rank correlation. In the males, there was a significant negative correlation of serum paraoxonase activity with total (P<0.05) and LDL (P<0.01) cholesterol levels, and positive correlation with HDL cholesterol and Apo A‐II levels (P< 0.05). Serum paraoxonase activity had a high positive correlation with serum triglyceride levels in both sexes (P< 0.001). Serum ApoB level had a positive correlation with the enzyme activity only in females (P<0.01). The allelic effect of PON on these parameters was studied by multiple regression analysis. The high activity allele (PON*B) was associated with higher serum triglyceride level (P<0.001) and ApoB (P<0.001), while it had lowering influence on total cholesterol (P<0.05) and LDL cholesterol (P<0.005) in men. The average allelic effect of PON was found to be about 22% for serum triglycerides, 11% for LDL cholesterol, 14% for Apo A‐II and 19% for Apo B in the present study. This study suggests a possible significant role of serum paraoxonase alleles in the metabolism of serum lipids and apolipoproteins.

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.

Microinjection of human oocytes: a technique for severe oligoasthenoteratozoospermia *

Objective To determine outcome after microinjection with very poor quality sperm and after failed fertilization. Design Group 1, fresh oocytes from patients with very low sperm density and motility on the day of oocyte recovery; Group 2, 1-day-old oocytes that failed to fertilize. Setting All material was obtained from the National University Hospital. Patients One hundred and thirty-one from group 1; 35 from group 2. Interventions Sperm was injected subzonally or directly into the ooplasm. Main Outcome Measure Normal and abnormal fertilization and pregnancy. Results Subzonal transfer was done on 771 oocytes in group 1 and 188 oocytes in group 2. Multiple sperm were transferred [mean of 6.6 for group 1 and 7.3 for group 2]. Monospermic fertilization occurred in 16.6% and 14.9%, respectively. Polyspermy and parthenogenetic activation were low at 2.3% and 2.8%, respectively. Five pregnancies were obtained, but only one delivered. Ooplasmic injection (single sperm heads) was done in 38 oocytes from three patients with extremely severe oligozoospermia; only four two-pronuclear zygotes were obtained and replaced into two patients, without any resulting pregnancy. Conclusions Subzonal transfer may be a viable technique for patients with severe sperm problems.

A new molecular variant of luteinizing hormone associated with female infertility

OBJECTIVE To investigate whether the newly described G1502 to A1502 mutation in exon 3 of the LH beta-subunit gene, causing the amino acid substitution of Ser102 for Gly102, is related to female infertility. DESIGN Screening of fertile and infertile women for the G1502 to A1502 mutation in the LH beta-subunit gene. SETTING Clinics and laboratories of the National University Hospital obstetrics and gynecology department, Singapore. PATIENT(S) Two hundred twelve healthy fertile women; 40 infertile women with menstrual disorders, polycystic ovary syndrome, and endometriosis; and 12 women with idiopathic infertility. INTERVENTION(S) Exon 3 of the LH beta-subunit gene was analyzed using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-mediated direct DNA sequencing. MAIN OUTCOME MEASURE(S) The PCR products of patients were analyzed by RFLP, and the results were compared with those of fertile controls. DNA sequencing radiographs were compared between two mutation-bearing patients and four controls. RESULT(S) The mutation was identified in only two infertile women with endometriosis; other women studied were found to be negative for this mutation. CONCLUSION(S) The missense mutation in the LH beta-subunit gene may be implicated in female infertility, possibly endometriosis-associated infertility in some women.

Micromanipulation: its relevance to human in vitro fertilization

Micromanipulation has been practiced for almost a century in invertebrates and lower animals. But in the past 2 decades, many micromanipulation procedures were initiated with the mouse embryo, including removal of the zona pellucida, dissociation of blastomeres, aggregation of denuded embryos and blastomeres, injection of single or aggregated cells into the cavity of early blastocysts, and insertion of isolated blastomeres into foreign zona pellucidae. Micromanipulation in the human will probably be limited to the following: (1) diagnosis and correction of genetic diseases and chromosomal disorders, and (2) assisting fertilization in severe oligozoospermia or motility problems of the spermatozoa

Implanon® contraceptive implants: effects on carbohydrate metabolism

The objective of the study was to assess the possible differences in effects of Implanon and Norplant implants on carbohydrate metabolism. This is a 2-year open randomized study of 80 implant (Implanon and Norplant) acceptors. Oral glucose tolerance test (OGTT) was performed before implant insertion and at 6, 12, and 24 months after implant insertion. Glycosylated hemoglobin A(1)C was measured in fasting samples and plasma samples during OGTT were tested for glucose and insulin levels. There was a significant increase in the area under the curve for both glucose and insulin during OGTT within each group with increasing duration of use. However, there was no significant change in the fasting plasma glucose values. There was no significant difference in the carbohydrate parameters between the two groups during implant use, except for a minimal but statistically significant rise in fasting glycosylated hemoglobin A(1)C levels at 24 months in the Implanon group. Both implants appear to induce mild insulin resistance but no significant change in serum glucose levels. These alterations in carbohydrate metabolism should have no clinical significance in healthy women.

USE OF BENZODIAZEPINES IN THE MANAGEMENT OF ECLAMPSIA

A GREAT variety of sedatives have been used in the management of eclampsia. At the Kandang Kerbau Hospital, the lytic “cocktail” and thiopentone sodium have been chiefly used (Hudson and Seah, 1956; Sheares, 1957); magnesium sulphate and promazine (Sparine) have also been used with favourable but inconsistent results. In 1965 good results for both mother and infant were obtained with Librium (also known as chlordiazepoxide . . . chemical formula 7-chloro-2-methylamino-5-phenyl-3H-l, 4-benzodiazepine 4-oxide) in the management of severe cases of pre-eclampsia and eclampsia (Ratnam et al., 1966). Very large doses were used with safety (Lean et al., 1968). The same impressions were recorded with the newer benzodiazepine derivative, Valium (also known as diazepam . . . chemical formula 7-chloro-1, 3-dihydro-lmethyl-5-phenyl-2H-1, 4-benzodiazepine-2-one) The benzodiazepines are the more recent psychotropic drugs which act mainly by depressing the reticular activating system and the basal ganglia of the brain. They do these without depressing the vital medullary centres. Besides its effects in counteracting anxiety (Smith, 1960; Iborra, 1963), Librium is an autonomic stabilizer and has sedative and hypnotic effects (Jenny, 1963). It causes muscular relaxation, including that of the uterus. Its anti-convulsive properties (Del Valle, 1961 ; Kaim and Rosenstein, 1960) enhance its value in the control of eclampsia. Some favourable reports on the use of the benzodiazepines on a smaller number of cases of eclampsia and pre-eclampsia have been published (Berger and Cavanagh, 1963; Leinzinger, 1964; Baumgarten, 1963 ; Kalkschmid, 1964; Gilbert, 1961). Gilbert was the first to report the successful use of Librium in a case of eclampsia. A favourable report on the use of Librium on 8 cases of eclampsia and 92 cases of severe pre-eclampsia has already been published (Ratnam et al., 1966).