Mental health and behavioural problems in adolescents conceived after ART.

STUDY QUESTION: Does mental health and behaviour differ between those conceived with and those conceived without ART? SUMMARY ANSWER: Our study observed less externalizing behaviour (delinquent/aggressive), and more parent-reported internalizing behaviour, as well as more (clinical) depression at age 14 years, in adolescents conceived after ART compared to their non-ART counterparts. WHAT IS KNOWN ALREADY: Health outcomes of ART-conceived offspring may differ from those conceived without ART, and previous studies have reported differences in behaviour and mental health, particularly in childhood. STUDY DESIGN, SIZE, DURATION: The Growing Up Healthy Study (GUHS) is a prospective cohort study, investigating the long-term health of offspring conceived after ART (aged 14, 17 and 20 years), in the two operational fertility clinics in Western Australia 1991-2001 (n = 303). Their long-term health outcomes were compared to those of offspring conceived without ART from the Raine Study Generation 2 (Gen2) born 1989-1991 (n = 2868). Both cohorts are representative of the local adolescent population. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mental health parameters and behaviour were assessed at ages 14 and 17 years, through the parent completed 'Child Behaviour Checklist' (CBCL; ART versus non-ART: age 14 years: N = 150 versus N = 1781, age 17 years: N = 160 versus N = 1351), and the adolescent completed equivalent 'Youth Self-Report' (YSR; age 14 years: by N = 151 versus N = 1557, age 17 years: N = 161 and N = 1232). Both tools generate a T-score (standardized for age and sex) for internalizing (withdrawn, somatic complaints, anxious/depressed), externalizing (delinquent/aggressive behaviour) and total behaviour. Adolescents also completed the 'Beck Depression Inventory for Youth' (BDI-Y; age 14 years: N = 151 versus N = 1563, age 17 years: N = 161 versus N = 1219). Higher scores indicate poorer mental health and behaviour on all the above tools. Parent-reported doctor-diagnosed conditions (anxiety, behavioural problems, attention problems and depression) were also univariately compared between the cohorts. In addition, univariate comparisons were conducted between the GUHS adolescents and Gen2 adolescents born to subfertile parents (time to pregnancy >12 months), as well as between offspring born to subfertile versus fertile parents within the Gen2 cohort. A subgroup analysis excluding offspring born preterm (<37 weeks' gestation) or at low birthweight (<2500 g) was also performed. Generalized estimating equations that account for correlated familial data were adjusted for the following covariates: non-singleton, primiparity, primary caregiver smoking, family financial problems, socio-economic status and both maternal and paternal ages at conception. MAIN RESULTS AND THE ROLE OF CHANCE: At both 14 and 17 years of age, ART versus non-ART-conceived adolescents reported lower mean T-scores for externalizing problems (age 14 years: 49 versus 51, P = 0.045, age 17 years: 49 versus 52, P < 0.001). A similar effect was reported by parents, although not significant (age 14 years: P = 0.293, age 17 years: P = 0.148). Fewer ART-conceived adolescents reported a T-score above the clinical cut-off for externalizing behaviour (≥60; age 14 years: 7.3% versus 16.3%, P = 0.003, age 17 years: 8.1% versus 19.7%, P < 0.001). At both ages, no differences in internalizing behaviour were reported by adolescents (age 14 years: P = 0.218, age 17 years: P = 0.717); however, higher mean scores were reported by parents of the ART-conceived adolescents than by parents of the non-ART conceived adolescents (age 14 years: 51 versus 48, P = 0.027, age 17 years: 50 versus 46, P < 0.001). No differences in internalizing behaviour above the clinical cut-off (T-score ≥ 60) were observed. At age 17 years, parents who conceived through ART reported higher total behaviour scores than those parents who conceived without ART (48 versus 45, P = 0.002). At age 14 years, ART versus non-ART-conceived adolescents reported significantly higher mean scores on the BDI-Y (9 versus 6, P = 0.005); a higher percentage of adolescents with a score indicating clinical depression (≥17; 12.6% versus 8.5%, aOR 2.37 (1.18-4.77), P = 0.016), as well as more moderate/severe depression (≥21; 9.3% versus 4.0%, P = 0.009). At age 17 years, no differences were reported on the BDI-Y. There was also a higher percentage of parent-reported doctor-diagnosed anxiety in the ART cohort (age 14 years: 8.6% versus 3.5%, P = 0.002, at age 17 years: 12.0% versus 4.5%, P < 0.001). Removing adolescents born preterm or at low birthweight did not alter the above results. Comparing outcomes between GUHS adolescents and Gen2 adolescents born to subfertile parents, as well as between those born to subfertile versus fertile parents within Gen2, did not alter results for CBCL and YSR outcomes. Those born to subfertile parents showed higher rates of clinical depression than those born to fertile parents at age 14 years (13.7% versus 6.9%, P = 0.035). LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study is the time difference between the GUHS and Gen2 assessments. Even though we have adjusted for covariates, additional socio-economic and lifestyle factors affecting behaviour and mental well-being could have changed. We were unable to differentiate between different types of ART (e.g. IVF versus ICSI), owing to the low number of ICSI cycles at the time of study. Fertility sub-analyses need to be replicated in larger cohorts to increase power, potentially using siblingship designs. Lastly, selection bias may be present. WIDER IMPLICATIONS OF THE FINDINGS: The reported lower prevalence of externalizing behaviour (delinquent/aggressive), and higher prevalence of internalizing behaviour, as well as more (clinical) depression at age 14 years, in ART versus non-ART-conceived adolescents, is in line with some previous studies, mostly conducted in childhood. It is reassuring that differences in the rates of depression were not observed at age 17 years, however, these findings require replication. As the use of ART is common, and mental health disorders are increasing, knowledge about a potential association is important for parents and healthcare providers alike. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by an NHMRC Grant (Hart et al., ID 1042269). R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director of PIVET Medical Centre, Perth, Western Australia. TRIAL REGISTRATION NUMBER: N/A.

Offspring conceived through ART have normal thyroid function in adolescence and as young adults.

STUDY QUESTION: Are there differences in thyroid function between adolescents and young adults conceived with and without ART? SUMMARY ANSWER: This study demonstrated no evidence of clinically relevant differences in thyroid function between adolescents and young adults conceived with and without ART. WHAT IS KNOWN ALREADY: Studies to date have reported an increase in subclinical hypothyroidism in offspring conceived after ART. It has been suggested that the increase in maternal estrogen (E2) after fresh embryo transfers could affect thyroid function of the offspring. Suboptimal thyroid function at a young age can cause irreversible damage to the central nervous system, which makes early detection and correct treatment essential. STUDY DESIGN, SIZE, DURATION: The Growing Up Healthy Study (GUHS) is a prospective cohort study, which aimed to recruit all adolescents born after conception with ART between 1991 and 2001 in the study area. The included participants (n = 303, aged 13-20 years) completed various health assessments. Depending on the age at enrolment, participants completed thyroid assessments at the 14- or 20-year follow-up. The outcomes of these replicated thyroid assessments were compared to those of participants conceived without ART from the Raine Study Generation 2 (Gen2). The Gen2 participants (n = 2868) were born between 1989 and 1992 and have been recognized to be representative of the local population. PARTICIPANTS/MATERIALS, SETTING, METHODS: Thyroid function assessments were compared between n = 134 GUHS and n = 1359 Gen2 adolescents at age 14 years and between n = 47 GUHS and n = 914 Gen2 young adults at age 20 years. The following mean thyroid hormone concentrations were compared between the cohorts: thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4) and thyroid peroxidase antibodies (TPOAb). The prevalence of the following thyroid hormone profiles, based on individual thyroid hormone concentrations, was compared: euthyroidism, subclinical and overt hypo- and hyperthyroidism and thyroid autoimmunity. Outcomes were compared between the cohorts, and univariately between fresh embryo transfers (ET) and frozen ET (FET) within the GUHS. The correlation between maternal peak E2 concentrations (pE2) and fT4 was assessed within the GUHS. MAIN RESULTS AND THE ROLE OF CHANCE: All mean thyroid function outcomes fell within the normal range. At both ages, we report no differences in TSH concentrations. At age 14 years, lower fT3 concentrations (4.80 versus 5.35 pmol/L, P < 0.001) and higher fT4 concentrations (12.76 versus 12.19 pmol/L, P < 0.001) were detected in the GUHS adolescents compared to Gen2 adolescents. At age 20 years, higher fT3 and fT4 concentrations were reported in GUHS adolescents (4.91 versus 4.63 pmol/L, P = 0.012; 13.43 versus 12.45 pmol/L, P < 0.001, respectively) compared to Gen2 participants. No differences in the prevalence of subclinical and overt hypo- and hyperthyroidism or thyroid autoimmunity were demonstrated between the cohorts at age 14 and 20 years. Thyroid function did not differ between ET and FET, and no correlation between pE2 and fT4 was reported. LIMITATIONS, REASONS FOR CAUTION: The observational nature of the study limits the ability to prove causation. Furthermore, the comparison of ET and FET offspring at age 20 years may be lacking power. We were unable to differentiate between different types of ART (e.g. IVF versus ICSI) owing to the low number of ICSI cycles at the time of study. As ART laboratory and clinic data were collected contemporaneously with the time of treatment, no other data pertaining to the ART cycles were sought retrospectively; hence, some factors could not be accounted for. WIDER IMPLICATIONS OF THE FINDINGS: This study does not support previous findings of clinically relevant differences in thyroid function when comparing a cohort of adolescents conceived after ART to counterparts conceived without ART. The minor differences detected in fT3 and fT4 were considered not biologically relevant. Although these findings appear reassuring, they warrant reinvestigation in adulthood. STUDY FUNDING/COMPETING INTERESTS: This project was funded by an NHMRC Grant (Hart et al., ID 1042269). R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director and a shareholder of PIVET Medical Centre, Perth, Western Australia. TRIAL REGISTRATION NUMBER: N/A.

Comparison of the cardiometabolic profiles of adolescents conceived through ART with those of a non-ART cohort.

STUDY QUESTION: Is the cardiometabolic health of adolescents conceived through ART worse than that of their counterparts conceived without ART? SUMMARY ANSWER: The majority of cardiometabolic and vascular health parameters of adolescents conceived through ART are similar or more favourable, than those of their counterparts of similar age and conceived without ART. WHAT IS KNOWN ALREADY: It has been proposed that the cardiometabolic health of offspring conceived with ART may be unfavourable compared to that of their counterparts conceived without ART. The literature pertaining to cardiometabolic health of offspring conceived after ART is contradictory, but generally suggests unfavourable cardiometabolic health parameters, such as an increase in blood pressure (BP), vascular dysfunction and adiposity, as well as unfavourable glucose and lipid profiles. With over 8 million children and adults born through ART worldwide, it is important to investigate whether these early signs of adverse cardiometabolic differences persist into adolescence and beyond. STUDY DESIGN, SIZE, DURATION: The Growing Up Healthy Study (GUHS) is a prospective cohort study that recruited 303 adolescents and young adults conceived after ART (aged 13-21 years) and born between 1991 and 2001 in Western Australia. Their health parameters, including cardiometabolic factors, were assessed and compared with counterparts from the Raine Study Generation 2 (Gen2). The 2868 Gen2 participants were born 1989-1992 and are representative of the Western Australian adolescent population. At ∼17 years of age (2013-2017), 163 GUHS participants replicated assessments previously completed by Gen2 at a similar age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cardiometabolic parameters were compared between a total of 163 GUHS and 1457 Gen2 adolescents. Separate male (GUHS n = 81, Gen2 n = 735) and female (GUHS n = 82, Gen2 n = 722) analyses were conducted. Assessments consisted of a detailed questionnaire including health, lifestyle and demographic parameters, anthropometric assessments (height, weight, BMI, waist circumference and skinfold thickness), fasting serum biochemistry, arterial stiffness and BP (assessed using applanation tonometry). Abdominal ultrasonography was used to assess the presence and severity of hepatic steatosis, and thickness of abdominal fat compartments. Non-alcoholic fatty liver disease (NAFLD) was diagnosed if there was sonographic fatty liver in the absence of significant alcohol consumption. Chi2, Fisher's exact and Mann-Whitney U tests, performed in SPSS V25, examined cohort differences and generalized estimating equations adjusted for the following covariates: singleton vs non-singleton pregnancy, birthweight (z-score), gestational age, BMI, smoking, alcohol consumption in the past 6 months and parent cardiovascular status. Arterial stiffness measures and waist circumference were additionally adjusted for height, and female analyses were additionally adjusted for use of oral contraceptives in the preceding 6 months. MAIN RESULTS AND THE ROLE OF CHANCE: In adjusted analyses, GUHS females had a lower BMI (22.1 vs 23.3 kg/m2, P = 0.014), and thinner skinfolds (triceps, subscapular, mid-abdominal; 16.9 vs 18.7 mm, P = 0.021, 13.4 vs 15.0 mm, P = 0.027, 19.7 vs 23.2 mm, P < 0.001, respectively), whereas males were not significantly different. Waist circumference was lower in GUHS adolescents (males: 78.1 vs 81.3 cm, P = 0.008, females: 76.7 vs 83.3 cm, P = 0.007). There were no significant differences between the two groups in glucose, insulin, homeostatic model assessment for insulin resistance, low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC), alanine aminotransferase and high-sensitivity C-reactive protein in both sexes. In females, serum triglycerides were lower in GUHS adolescents (1.0 vs 1.2 mmol/l, P = 0.029). GUHS males had higher serum HDL-C (1.1 vs 1.0 mmol/l, P = 0.004) and a lower TC/HDL-C ratio (3.2 vs 3.6, P = 0.036). There were no significant differences in the prevalence of NAFLD or steatosis severity scores between the cohorts in males and females. GUHS females had less subcutaneous adipose tissue (9.4 vs 17.9 mm, P < 0.001), whereas GUHS males had greater visceral adipose thickness (44.7 vs 36.3 mm, P < 0.001). There was no significant difference in pre-peritoneal adipose thickness. Pulse wave velocity was lower in GUHS males (5.8 vs 6.3 m/s, P < 0.001) and heart rate corrected augmentation index was lower in GUHS females (-8.4 vs -2.7%, P = 0.048). There were no significant differences in BP or heart rate in males or females between the two groups. LIMITATIONS, REASONS FOR CAUTION: Despite the substantial study size and the unique study design of the ART cohort, we were unable to differentiate between different types of ART, due to the low number of ICSI cycles (e.g. IVF vs ICSI), draw definite conclusions, or relate the outcomes to the cause of infertility. Considering the differences in time points when both cohorts were studied, external factors could have changed, which could not be accounted for. Given the observational nature of this study, causation cannot be proven. WIDER IMPLICATIONS OF THE FINDINGS: Contrary to our hypothesis and previous findings focussing mainly on childhood, this study reports mostly similar or favourable cardiometabolic markers in adolescents conceived with ART compared to those conceived without ART. The greater visceral adipose thickness, particularly present in males, requires further investigation. While these findings are generally reassuring, future well-designed and appropriately powered studies are required to definitively address the issue of cardiometabolic health in ART adults. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NHMRC project grant number 1042269 and R.J.H. received education grant funding support from Ferring Pharmaceuticals. R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director of PIVET Medical Centre, Perth, Western Australia. TRIAL REGISTRATION NUMBER: N/A.

DNA methylation patterns within whole blood of adolescents born from assisted reproductive technology are not different from adolescents born from natural conception.

STUDY QUESTION: Do the epigenome-wide DNA methylation profiles of adolescents born from ART differ from the epigenome of naturally conceived counterparts? SUMMARY ANSWER: No significant differences in the DNA methylation profiles of adolescents born from ART [IVF or ICSI] were observed when compared to their naturally conceived, similar aged counterparts. WHAT IS KNOWN ALREADY: Short-term and longer-term studies have investigated the general health outcomes of children born from IVF treatment, albeit without common agreement as to the cause and underlying mechanisms of these adverse health findings. Growing evidence suggests that the reported adverse health outcomes in IVF-born offspring might have underlying epigenetic mechanisms. STUDY DESIGN, SIZE, DURATION: The Growing Up Healthy Study (GUHS) is a prospective study that recruited 303 adolescents and young adults, conceived through ART, to compare various long-term health outcomes and DNA methylation profiles with similar aged counterparts from Generation 2 from the Raine Study. GUHS assessments were conducted between 2013 and 2017. The effect of ART on DNA methylation levels of 231 adolescents mean age 15.96 ± 1.59 years (52.8% male) was compared to 1188 naturally conceived counterparts, 17.25 ± 0.58 years (50.9% male) from the Raine Study. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA methylation profiles from a subset of 231 adolescents (13-19.9 years) from the GUHS, generated using the Infinium Methylation Epic Bead Chip (EPIC) array were compared to 1188 profiles from the Raine Study previously measured using the Illumina 450K array. We conducted epigenome-wide association approach (EWAS) and tested for an association between the cohorts applying Firth's bias reduced logistic regression against the outcome of ART versus naturally conceived offspring. Additionally, within the GUHS cohort, we investigated differences in methylation status in fresh versus frozen embryo transfers, cause of infertility as well as IVF versus ICSI conceived offspring. Following the EWAS analysis we investigated nominally significant probes using Gene Set Enrichment Analysis (GSEA) to identify enriched biological pathways. Finally, within GUHS we compared four estimates (Horvath, Hanuum, PhenoAge [Levine], and skin Horvath) of epigenetic age and their correlation with chronological age. MAIN RESULTS AND THE ROLE OF CHANCE: Between the two cohorts, we did not identify any DNA methylation probes that reached a Bonferroni corrected P-value < 1.24E-0.7. When comparing IVF versus ICSI conceived adolescents within the GUHS cohort, after adjustment for participant age, sex, maternal smoking, multiple births, and batch effect, three methylation probes (cg15016734, cg26744878 and cg20233073) reached a Bonferroni correction of 6.31E-08. After correcting for cell count heterogeneity, two of the aforementioned probes remained significant and an additional two probes (cg 0331628 and cg 20235051) were identified. A general trend towards hypomethylation in the ICSI offspring was observed. All four measures of epigenetic age were highly correlated with chronological age and showed no evidence of accelerated epigenetic aging within their whole blood. LIMITATIONS, REASONS FOR CAUTION: The small sample size coupled with the use of whole blood, where epigenetic differences may occur in other tissue. This was corrected by the utilized statistical method that accounts for imbalanced sample size between groups and adjusting for cell count heterogeneity. Only a small portion of the methylome was analysed and rare individual differences may be missed. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide further reassurance that the effects of the ART manipulations occurring during early embryogenesis, existing in the neonatal period are indeed of a transient nature and do not persist into adolescence. However, we have not excluded that alternative epigenetic mechanisms may be at play. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NHMRC project Grant no. 1042269 and R.J.H. received funding support from Ferring Pharmaceuticals Pty Ltd. R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from Merck Sharp & Dohme Corp.- Australia, Merck-Serono Australia Pty Ltd and Ferring Pharmaceuticals Pty Ltd. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director of PIVET Medical Centre, Perth, Western Australia. The remaining authors have no conflicts of interest.

A direct action for GH in improvement of oocyte quality in poor-responder patients.

Declining female fecundity at later age and the increasing tendency for women to delay childbirth have lead to a drastic rise in the number of women seeking assisted reproductive technology. Many women fail to respond adequately to standard ovarian stimulation regimens, raising a significant therapeutic challenge. Recently, we have demonstrated that the administration of GH, as an adjunct to ovarian stimulation, has improved the clinical outcomes by enhancing the oocyte quality. However, the mechanism(s) by which GH facilitated this improvement is yet to be understood. This study aimed to determine these potential mechanism(s) through the use of immunofluorescent localisation of GH receptors (GHRs) on the human oocyte and unbiased computer-based quantification to assess and compare oocyte quality between women of varying ages, with or without GH treatment. This study demonstrates for the first time, the presence of GHRs on the human oocyte. The oocytes retrieved from older women showed significant decrease in the expression of GHRs and amount of functional mitochondria when compared with those from younger patients. More interestingly, when older patients were treated with GH, a significant increase in functional mitochondria was observed in their oocytes. We conclude that GH exerts a direct mode of action, enabling the improvement of oocyte quality observed in our previous study, via the upregulation of its own receptors and enhancement of mitochondrial activity. This result, together with recent observations, provides scientific evidence in support of the use of GH supplementation for the clinical management of poor ovarian response.

Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment.

Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation.

Changes in total and free concentrations of steroid hormones in the plasma of women throughout pregnancy: effects of medroxyprogesterone acetate in the first trimester.

The total (protein-bound plus free) and free concentrations of progesterone, oestradiol-17 beta and cortisol were measured serially throughout pregnancy in the plasma of two groups of women whose pregnancies went to term. Group A (n = 53) experienced an uneventful low-risk pregnancy with a spontaneous abortion rate of 8.6%. Women in group B (n = 22) were treated orally with medroxyprogesterone acetate (MPA; 80-120 mg daily) until 18 weeks gestation for threatened abortion within the first 6 weeks of pregnancy. In both groups of women the proportion of each hormone circulating in the free or unbound form remained constant despite the overall increases with occurred in total circulating hormone concentrations as pregnancy progressed. The steroid hormonal profiles in the first half of pregnancy were similar in both groups of women. However, from weeks 20 to 40 total and free progesterone concentrations were significantly (P less than 0.05 in each case) higher in group B compared with group A. Conversely, total and free oestradiol-17 beta concentrations were lower (P less than 0.005 and P less than 0.01 respectively) in group B. At this stage it is not known if these differences were attributable to the administration of MPA to women in group B or to altered placental steroidogenesis as a result of earlier uterine bleeding.

Medroxyprogesterone acetate does not perturb the profile of steroid metabolites in urine during pregnancy.

The plasma concentrations of medroxyprogesterone acetate (MPA) in 14 women administered the progestagen for threatened abortion during the first 6 weeks of pregnancy were measured by specific radioimmunoassay. Treatment (52 nmol orally every 6 h) was continued to 18 weeks of gestation. The mean plasma concentration of MPA rose rapidly during day 1 of treatment to 14.1 +/- 1.84 nmol/l. It reached 21.5 +/- 2.3 nmol/l by 7 days and subsequently stabilized at around 26.8 +/- 5.0 nmol/l by the end of week 2. Urinary steroid profiles were determined by gas-liquid chromatography and mass spectrometry for six of the MPA-treated women and compared with those of six untreated women of similar gestational age. No differences were detected between the two groups of women, suggesting that the administration of MPA during pregnancy did not alter qualitatively or quantitatively the metabolism and excretion into urine of progesterone and oestrogens.

The management of oligospermic infertility by in vitro fertilization.

The fertilization rates of mature preovulatory oocytes aspirated from 156 women treated by in vitro fertilization were analyzed as a function of spermatozoal density and motility and the findings were correlated with the category of infertility (chronic oligospermia, tubal disease, endometriosis, serum antibodies to spermatozoa in the female, and unexplained infertility). Overall reduced fertilization rates were found in all cases if the semen sample presented on the day of fertilization demonstrated less than or equal to 5 million motile spermatozoa per milliliter, less than 40% motile forms, or the combined findings of less than 20 million per ml and less than or equal to 60% motile forms. Where the husband was known to have chronic oligospermia, reduced fertilization was found if the semen on the day of fertilization contained less than 20 million spermatozoa per ml and less than 12 million motile spermatozoa per milliliter. For cases of unexplained infertility, a poor fertilization rate was noted if the semen demonstrated less than 60% progressively motile forms regardless of the overall spermatozoal density, implying that a proportion of unexplained infertility is due to a disorder of spermatozoa reflected by reduced motility. Pregnancies were achieved in 5 of 26 cases with chronic oligospermia, including 2 where oligospermia was very pronounced (less than or equal to 5 million motile spermatozoa per milliliter).

Combined pregnancy after gonadotropin therapy.

Three cases of combined pregnancy are described after gonadotropin therapy; two cases after human pituitary gonadotropin and one after human menopausal gonadotropin administration. In each case the intrauterine gestation was a multiple pregnancy. After salpingectomy, two of the women have proceeded to the delivery of healthy infants; the third woman aborted. In each case the gonadotropin stimulation regimen was ceased at the appropriate stage when the estriol excretion was between 60 and 125 micrograms/day, but the subsequent rate of rise of estriol was 2.3- to 3.2-fold during the coasting phase before the human chorionic gonadotropin trigger when the estriol excretion rate was 140 to 350 micrograms/day.

The treatment of infertility associated with endometriosis by in vitro fertilization.

In vitro fertilization and embryo transfer (IVF-ET) was performed on women with tubal disease only (28 women, 40 cycles), endometriosis grade I (15 women, 24 cycles), grade II (25 women, 37 cycles), grade III (26 women, 36 cycles), or grade IV (31 women, 52 cycles). Rates of oocyte recovery and fertilization were not affected by the presence of endometriosis. Pregnancy rates per cycle were similar to that of the tubal group (18%) for women with grade I (13%) or grade II (14%) endometriosis, lower in women with grade III endometriosis (6%) and significantly reduced in women with grade IV (2%, P less than 0.05).

Failure of human oocyte release at ovulation.

Among 150 patients admitted for ovum aspiration, in vitro fertilization, and embryo transfer in Perth, Western Australia, 14 were found to have had at least one ovulated follicle at the time of laparoscopy. Based upon ultrasound estimation of follicle diameter 24 hours previously, ovulation occurred in 6 of 22 follicles less than 1.7 cm in mean diameter and in 16 of 19 follicles greater than 1.7 cm in mean diameter. When the ruptured follicles were flushed with heparinized fertilization medium, oocytes were collected in 13 of the 22 dispersed follicles. Twelve oocytes developed pronuclei 16 hours after the addition of spermatozoa, and 11 cleaved to the 4-cell stage within 44 hours of insemination. Oocytes were recovered from 6 of 11 patients stimulated with clomiphene alone and from all 3 patients stimulated with clomiphene supplemented with human menopausal gonadotropin. These observations suggest that oocyte release and follicle rupture are not necessarily synonymous events and that the incidence of retained oocytes in ovulated follicles following stimulation with clomiphene or clomiphene plus human menopausal gonadotropin could be on the order of 60%.

The relative chance of pregnancy following tubal or uterine transfer procedures.

In a 7-month study period, a total of 113 pregnancies were generated in 380 women (30%) undergoing transfers in one of four assisted conception procedures: gamete intrafallopian transfer (GIFT), pronuclear stage tubal transfer (PROST), tubal embryo stage transfer (TEST), and in vitro fertilization and embryo transfer (IVF-ET). It was shown that both the pregnancy rate per transfer procedure and the number of pregnancy sacs arising per embryo transferred were significantly higher among the groups having tubal transfer (P less than 0.001). There were no significant differences in the pregnancy or implantation rates among the three groups having tubal transfer procedures when the GIFT results were adjusted for a 72% fertilization rate noted in the combined IVF-ET and PROST groups. Early pregnancy wastage showed a similar pattern among the four groups and, overall, 67% of pregnancies advanced beyond 20 weeks.

Embryo transfer technique as a cause of ectopic pregnancies in in vitro fertilization.

Against an overall risk of around 2% ectopic pregnancies in in vitro fertilization programs around the world, we report an incidence more than three times greater (5 tubal ectopic pregnancies in 80 clinical in vitro fertilization pregnancies). Of two techniques used for embryo transfers, one produced a significantly higher risk for ectopic pregnancy (P less than 0.05). Four of the ectopic pregnancies occurred in a small group of 24 patients in whom the embryo transfer technique attempted to deliver the embryos at the uterine fundus (mean distance of catheter insertion 62.9 +/- 7.9 mm from the external cervical os). Only one ectopic pregnancy occurred in 56 patients whose embryos were transferred to a standard, generally midcavity position. It is concluded that the delivery catheter need be inserted only 55 mm as a routine and less in patients with a shortened cervix or with the hypoplastic uterus usually encountered in women with primary ovarian failure who have ovum or embryo donation. In such cases an ultrasonic measurement of length may indicate that a shorter transfer distance is required.

Hormonal profiles and embryo quality in women with severe endometriosis treated by in vitro fertilization and embryo transfer.

A study was undertaken comparing the outcomes of 30 women with infertility due to untreated severe (grade IV) pelvic endometriosis with a comparable series of 28 women whose infertility was caused solely by irreversible tubal disease. There were no significant differences in either the follicular phase or luteal phase hormonal profiles of estradiol and progesterone, but there was a significantly reduced pregnancy rate in those women with severe endometriosis. In part, this was due to the recovery of fewer oocytes from the endometriosis patients (P less than 0.001) despite the fact that the peak estradiol levels and ovarian accessibility were similar in the two groups. However, there were no significant differences in the proportion of oocytes that fertilized or the number that demonstrated normal embryo growth and high-grade embryo quality. There also appears to be an implantation inhibitory factor in patients with severe endometriosis as the pregnancy rate/embryo transferred and number of gestational sacs identified/embryo transferred were significantly reduced (P less than 0.05).

Evaluation of luteal support therapy in a randomized controlled study within a gamete intrafallopian transfer program.

A randomized controlled study of luteal support therapy (using intramuscular injections of progesterone and/or human chorionic gonadotropin) was conducted in a trial designed to minimize variables that might adversely affect the change of pregnancy. After applying rigid selection criteria, 207 women were recruited into one of four groups. Mathematical modeling was applied to the results to determine if there were degrees of improvement in uterine receptivity relative to various grades of embryo quality ("E" factor). Although the trial size was insufficient to enable the detection of significant improvements in the pregnancy rates that ranged from 27.5% for non-treatment to 41.2% for those receiving combined treatment, the birth rates were significantly better with luteal support (11.8% versus 29.4%). Similarly, the overall implantation rate just failed to reach statistical significance for luteal support, but the ongoing implantations were significantly better (3.6% versus 9.0%). Data modeling indicated that luteal support, particularly with the combined regimen, could improve the ongoing implantation rate by up to 2.5-fold when the E factor was poorest.

Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study.

OBJECTIVE: To assess the utility of the acrosome reaction (AR) to ionophore challenge test in determining the sperm treatment protocols for patients undergoing assisted reproduction. DESIGN, SETTING, PATIENTS: One hundred twenty-one couples undergoing an IVF-ET or GIFT procedure from January to July 1992 were included in this prospective study. All cases had a preliminary semen analysis within the previous 3 months and an AR to ionophore challenge test was carried out unless an acceptable fertilization rate occurred on previous IVF. For those patients whose AR to ionophore challenge score was below the accepted fertile range of > or = 10%, a second AR to ionophore challenge test was performed after exposure of sperm to the stimulant pentoxifylline. Couples then were managed by assisted reproduction with randomized allocation of oocytes for fertilization with a standard sperm preparation or with added sperm stimulants, either 3.6 mM pentoxifylline alone or combined with 3.0 mM 2-deoxyadenosine. The study was double-blind with neither the patients nor the embryologist knowing the AR to ionophore challenge result at the time of the IVF procedure. MAIN OUTCOME MEASURES: Data from the preliminary semen analyses and AR to ionophore challenge scores were correlated with the fertilization rates achieved using control and treated sperm preparations. The rates of total fertilization failure and the numbers of clinical pregnancies occurring in each subgroup were also recorded. RESULTS: All AR to ionophore challenge groups showed normal sperm counts except the groups with poor AR to ionophore challenge, which demonstrated reduced sperm counts. The group with normal AR to ionophore challenge scores or previous normal fertilization showed satisfactory fertilization rates with either control or treated sperm, although some individual cases showed reduced fertilization with treated sperm. The fertilization rate for the group with low AR to ionophore challenge scores improved significantly with pentoxifylline, and the benefit was greatest when this had been predicted from the AR to ionophore challenge studies. Cases with persisting poor AR to ionophore challenge despite pentoxifylline showed no significant improvement in fertilization rates with sperm exposed to either sperm stimulant regimens. Poor AR to ionophore challenge scores were also predictive of total fertilization failure, but this problem was reduced by sperm stimulation. The AR to ionophore challenge score at 10% cutoff level showed optimal levels of sensitivity (82.1%), highest negative predictive value (82.1%), and lowest false negative rate (17.9%). CONCLUSIONS: The AR to ionophore challenge test is useful in the assessment and management of the male factor in assisted reproduction. It can be used to identify the majority of cases who will benefit from the use of sperm stimulants.

Influence of pentoxifylline in severe male factor infertility.

Two in vitro fertilization sperm preparation protocols using pentoxifylline (long and short exposure before insemination) were studied in 57 couples (61 cycles) with male factor infertility. For each cycle, oocytes were divided into two groups for insemination using either pentoxifylline-treated or control semen. Fertilization rates improved over controls in the short protocol (P less than 0.001) and fewer couples experienced fertilization failure (P = 0.02). Sixteen pregnancies ensued (30% per collection with the short protocol), and 4 were from cases with less than 1.0 X 10(6) progressively motile sperm count per milliliter, 1 being as low as 0.2 X 10(6) progressively motile count per milliliter. Seventeen healthy infants have now delivered and pregnancy wastage is not increased. Pentoxifylline is thus a useful sperm treatment for cases of male factor infertility.

Preliminary results using pentoxifylline in a pronuclear stage tubal transfer (PROST) program for severe male factor infertility.

In vitro trials with washed spermatozoa incubated in medium containing 1 mg/ml of the methyl xanthine phosphodiesterase inhibitor PF showed improved counts of total motile and total progressively motile spermatozoa in cases of oligospermia/asthenospermia. Application of this agent in a PROST program for a series of nine couples presenting for treatment with histories of failed fertilization in vitro resulted in five pregnancies (four singleton, one triplet) and the subsequent delivery of normal infants. The results warrant further evaluation of this sperm treatment for cases of severe male factor infertility.

Early luteal serum progesterone concentrations are higher in pregnancy cycles.

In a consecutive series of 167 patients reaching the stage of embryo transfer after in vitro fertilization and embryo transfer, 19 clinical pregnancies ensued. The serum progesterone (P) levels were significantly greater on the first and second (P less than 0.01) and third (P less than 0.05) postaspiration days for those who conceived. Higher circulating levels of P were achieved on days 1, 2, and 3 (P less than 0.05) by the daily injection of P, 50 mg in oil, given for 5 consecutive days, beginning immediately after follicle aspiration. Both pregnancy and nonpregnancy cycles demonstrated high circulating P levels, but the study implies that relatively higher levels are required for conception, and such levels can be achieved by the use of intramuscular P.