John A. Allan

IMMUNOPATHOGENESIS OF CHLAMYDIA TRACHOMATIS INFECTIONS IN WOMEN

OBJECTIVE To develop a model of pathogenesis by which Chlamydia trachomatis progresses from acute to chronic infection, and finally serious disease (salpingitis, tubal occlusion). DESIGN Review of current literature located through web-based Medline searches using key words: Chlamydia trachomatis, immunology, cytokines, heat shock protein, infertility. RESULT(S) Cell-mediated immune mechanisms appear to be critical in determining whether acute infection is resolved or progresses into chronicity with pathological outcome. What determines the particular immune pathway depends on a range of determinants-HLA subtype and human genetics, cytokine profile, infectious load, route of infection, and endocrinology. A clearer picture of the natural history of chlamydial pathology may assist in providing better predictors of those women who may go on to develop significant sequelae after infection. CONCLUSION(S) Predicting those who may develop serious disease, including infertility, may contribute to improved management of such persons during earlier stages of infection and assist in prevention.

Ureaplasma parvum and Ureaplasma urealyticum are detected in semen after washing before assisted reproductive technology procedures

OBJECTIVE To investigate the prevalence of ureaplasmas in semen and washed semen and to explore their effect on semen andrology variables. DESIGN Prospective study. SETTING In vitro fertilization (IVF) unit of a private hospital. PATIENT(S) Three hundred forty-three men participating in an assisted reproductive technology (ART) treatment cycle. MAIN OUTCOME MEASURE(S) The prevalence of ureaplasmas in semen and washed semen tested by culture, polymerase chain reaction assays, and indirect immunofluorescent antibody assays. RESULT(S) Ureaplasmas were detected in 73 of 343 (22%) semen samples and 29 of 343 (8.5%) washed semen samples. Ureaplasmas adherent to the surface of spermatozoa were demonstrated by indirect immunofluorescent antibody testing. Ureplasma parvum serovar 6 (36.6%) and U. urealyticum (30%) were the most prevalent isolates in washed semen. A comparison of the semen andrology variables of washed semen ureaplasma positive and negative groups demonstrated a lower proportion of nonmotile sperm in men ureaplasma positive for washed semen. CONCLUSION(S) Ureaplasmas are not always removed from semen by a standard ART washing procedure and can remain adherent to the surface of spermatozoa.

A new method for freezing testicular biopsy sperm: Three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule

OBJECTIVE To determine whether spermatozoa, located in the seminiferous tubules obtained by needle puncture testicular biopsy, could be cryopreserved successfully within the tubules and subsequently be used for in oocyte fertilization via intracytoplasmic sperm injection (ICSI) after the spermatozoa were removed from the thawed tubules. DESIGN Clinical case series. SETTING Private IVF unit. PATIENT(S) Six azoospermic patients (four obstructive, two maturation arrest). MAIN OUTCOME MEASURE(S) Survival rate of thawed spermatozoa, fertilization rate of oocytes after ICSI with spermatozoa extracted from thawed tubules and pregnancies. RESULT(S) All six patients had adequate motile spermatozoa extracted from the thawed tubule sections, and all patients achieved fertilization via ICSI with their partners eggs. The fertilization rate was 46%, compared with 56% obtained in other previous patient cycles using fresh testicular spermatozoa. Three pregnancies resulted from five ETs. CONCLUSION(S) Cryopreservation and subsequent thawing of seminiferous tubules proved to be a simple and successful method for storage of testicular spermatozoa, reducing the need for repetitive testicular biopsies and increasing the likelihood of sperm availability on the day of oocyte retrieval.

Human and Pathogen Factors Associated with Chlamydia trachomatis-Related Infertility in Women

SUMMARY Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen worldwide. Infection can result in serious reproductive pathologies, including pelvic inflammatory disease, ectopic pregnancy, and infertility, in women. However, the processes that result in these reproductive pathologies have not been well defined. Here we review the evidence for the human disease burden of these chlamydial reproductive pathologies. We then review human-based evidence that links Chlamydia with reproductive pathologies in women. We present data supporting the idea that host, immunological, epidemiological, and pathogen factors may all contribute to the development of infertility. Specifically, we review the existing evidence that host and pathogen genotypes, host hormone status, age of sexual debut, sexual behavior, coinfections, and repeat infections are all likely to be contributory factors in development of infertility. Pathogen factors such as infectious burden, treatment failure, and tissue tropisms or ascension capacity are also potential contributory factors. We present four possible processes of pathology development and how these processes are supported by the published data. We highlight the limitations of the evidence and propose future studies that could improve our understanding of how chlamydial infertility in women occurs and possible future interventions to reduce this disease burden.

Microbial colonization of follicular fluid: alterations in cytokine expression and adverse assisted reproduction technology outcomes

BACKGROUND Previous studies have measured cytokines expressed within follicular fluid and compared the profiles with the aetiology of infertility and/or successful or unsuccessful assisted reproduction technology (ART) outcomes. METHODS In this study, 71 paired follicular fluid and vaginal secretions collected from ART patients were cultured to detect microorganisms and tested for the presence of cytokines. Patient specimens were selected for assay based on two criteria: whether the follicular fluid specimen was colonized (with microorganisms prior to oocyte retrieval) or contaminated by vaginal flora and; the aetiology of infertility. Patients included fertile women (with infertile male partners; n = 18), women with endometriosis (n = 16) or polycystic ovary syndrome (PCOS, n = 14), or couples with a history of genital tract infection (n = 9) or idiopathic infertility (n = 14). RESULTS Microorganisms and cytokines were detected within all tested specimens. Colonizing microorganisms in follicular fluid were associated with: decreased fertilization rates for fertile women (P = 0.005), women with endometriosis (P = 0.0002) or PCOS (P = 0.002) compared with women whose follicular fluid was contaminated at the time of oocyte retrieval and with decreased pregnancy rates for couples with idiopathic infertility (P = 0.001). A single cytokine was discriminatory for women with an idiopathic aetiology of infertility (follicular fluid interleukin (IL)-18). Unique cytokine profiles were also associated with successful fertilization (IL-1α, IL-1β, IL-18 and vascular endothelial growth factor). CONCLUSIONS Follicular fluid is not sterile. Microorganisms colonizing follicular fluid and the ensuing cytokine response could be a further as yet unrecognized cause and/or predictor of adverse ART outcomes and infertility.

Microorganisms within Human Follicular Fluid: Effects on IVF

Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.

Interaction of microbiology and pathology in women undergoing investigations for infertility

BACKGROUND Cases of endometriosis with no tubal damage are associated with infertility, suggesting an immunological rather than mechanical barrier to reproduction. Laparoscopy and falloposcopy results of clinically asymptomatic women undergoing investigation of infertility were correlated with the outcomes of microbiological screening for Chlamydia trachomatis, Mycoplasma pneumoniae, Mycoplasma hominis, ureaplasma species, Neisseria gonorrhoeae, Neisseria meningitidis and Chlamydia pneumoniae. METHODS A total of 44 women presenting to a hospital IVF service for laparoscopic or laparoscopic/falloposcopic investigation of infertility provided endocervical swabs, fallopian tube washings, and peripheral whole blood for analysis. RESULTS Of these 44 women, 15.9% (7) showed evidence of C. trachomatis infection as detected by either PCR or EIA serology. Of these 7 women, 5 (71%) had no or mild endometriosis and 2 (29%) had moderate or severe endometriosis. Of the remaining 37 women who showed no evidence of chlamydial infection, 15 (40.5%) had no or mild endometriosis. CONCLUSION Women with infertility, but without severe endometriosis at laparoscopy, showed a trend towards tubal damage and a higher rate of previous C. trachomatis infection. Although not statistically significant, this trend would suggest that, where moderate to severe tubal damage is found to be the primary cause of infertility, C. trachomatis infection could be a likely cause for such tubal damage.

The protease inhibitor JO146 demonstrates a critical role for CtHtrA for Chlamydia trachomatis reversion from penicillin persistence

The Chlamydia trachomatis serine protease HtrA (CtHtrA) has recently been demonstrated to be essential during the replicative phase of the chlamydial developmental cycle. A chemical inhibition strategy (serine protease inhibitor JO146) was used to demonstrate this essential role and it was found that the chlamydial inclusions diminish in size and are lost from the cell after CtHtrA inhibition without formation of viable elementary bodies. The inhibitor (JO146) was used in this study to investigate the role of CtHtrA for penicillin persistence and heat stress conditions for Chlamydia trachomatis. JO146 addition during penicillin persistence resulted in only minor reductions (~1 log) in the final viable infectious yield after persistent Chlamydia were reverted from persistence. However, JO146 treatment during the reversion and recovery from penicillin persistence was completely lethal for Chlamydia trachomatis. JO146 was completely lethal when added either during heat stress conditions, or during the recovery from heat stress conditions. These data together indicate that CtHtrA has essential roles during some stress environments (heat shock), recovery from stress environments (heat shock and penicillin persistence), as well as the previously characterized essential role during the replicative phase of the chlamydial developmental cycle. Thus, CtHtrA is an essential protease with both replicative phase and stress condition functions for Chlamydia trachomatis.

Re: Case report: Pregnancy from intracytoplasmic injection of a frozen-thawed oocyte1

This publication presents the first Australian pregnancy resulting from intracytoplasmic injection (ICSI) of a frozenthawed oocyte. We wish to report that at the Wesley IVF Service we achieved a similar pregnancy in 1998. As the pregnancy did not result in a birth, it was not submitted for publication in the scientific journals. The patient was 32 years old, and due to religious reasons, the couple did not want embryos created in excess of the number to be transferred. They were offered IVF with ICSI for fertilisation of the requested three oocytes. The remaining eight oocytes in this cycle were cryopreserved using the same cryopresevation protocol as described in the paper. The patient became pregnant in the treatment cycle, but miscarried at 6 weeks. She subsequently came back 2 months later for a frozen embryo transfer intending to use the cryopreserved oocytes. She was placed on hormone replacement for the frozen embryo transfer cycle. Four oocytes were thawed and three survived. Fertilisation (2 PN) was confirmed for all oocytes using ICSI in human tubal fluid medium (HTF, Irvine Scientific, Santa Ana, CA, USA) and on day 3, one of each of four-cell, five-cell and seven-cell stage embryos were transferred into the uterus. The patient was diagnosed as pregnant and her β hCG level 19 days after the transfer was 1382 pmol/L. She started to bleed and unfortunately miscarried. So far in our IVF programme, we have thawed 36 oocytes in seven treatment cycles and 18 oocytes survived (50%) the thaw. Of these, six oocytes fertilised (33.3%) and the resulting embryos were transferred in three cycles giving the single positive pregnancy we described earlier. Oocyte freezing as discussed by Kan et al. is not as successful as embryo preservation. Researchers are attempting to improve cryosurvival of oocytes using modified techniques.

Hormone-dependent bacterial growth, persistence and biofilm formation--a pilot study investigating human follicular fluid collected during IVF cycles.

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.