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Reduced Serum Testosterone Levels in Infant Boys Conceived by Intracytoplasmic Sperm Injection

University Department of Growth and Reproduction (C.M.K., K.M.M., N.E.S., A.J.), and The Fertility Clinic (A.N.A., A.L.), Copenhagen University Hospital Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

Address all correspondence and requests for reprints to: Anders Juul, M.D., D.M.Sc., Ph.D., University Department of Growth and Reproduction GR, Rigshospitalet section 5064, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. E-mail: ajuul{at}rh.hosp.dk.

	Abstract

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Context: Concern has been raised for the health of the offspring conceived by assisted reproduction technologies. Basal reproductive hormones around 3 months of age reflect the pituitary-testicular axis, which is transiently active at this age.

Objectives: We tested the hypothesis that transmission of impaired testicular function from father to son could be detected at 3 months of age in boys conceived by intracytoplasmic sperm injection (ICSI), which is predominantly used in the management of male infertility.

Design: We conducted a longitudinal prospective cohort study, including 125 boys conceived by ICSI, 124 boys conceived by in vitro fertilization (IVF), and 933 naturally conceived (NC) boys.

Intervention: Anthropometrical measurements were performed at birth and at 3 months of age; 58, 67, and 64% of ICSI, IVF, and NC boys, respectively, had a blood sample taken at 3 months.

Main Outcome Measures: We measured serum levels of LH, FSH, SHBG, inhibin B, testosterone, as well as penile length.

Results: Serum testosterone levels were significantly lower in boys conceived by ICSI (2.4 nmol/liter; 0.2–4.9 nmol/liter) (median; 2.5th–97.5th percentiles) compared with NC boys (3.3 nmol/liter; 0.6–7.6 nmol/liter; P < 0.001), and the LH to testosterone ratio was increased (0.8; 0.2–7.9 vs. 0.5; 0.2–2.3, respectively; P = 0.001). Boys conceived by IVF because of female infertility factors had a normal serum testosterone and LH to testosterone ratio compared with controls. Adjusted analyses for confounders did not alter the results.

Conclusions: Our results point toward a subtle impairment of Leydig cell function in boys conceived by ICSI, possibly inherited from their fathers. The clinical significance of our findings is uncertain. However, our findings should raise concern because ICSI is increasingly used to overcome male infertility.

	Introduction

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ASSISTED REPRODUCTION technologies (ART) are increasingly used worldwide. In 2002, the highest rate of European children born after ART was found in Denmark, where 1.5% of all newborns were conceived by intracytoplasmic sperm injection (ICSI) and 2.3% by in vitro fertilization (IVF) (1). ICSI is used mainly for male infertility, and concern has been raised that boys conceived by ICSI may inherit an impaired testicular function from their father. Several authors (2, 3, 4, 5) have reported on familial clustering of male infertility problems. Little attention has been paid to the fact that some infertile men have impaired Leydig cell function and lower serum testosterone (6), and Leydig cell function has not been evaluated in ICSI offspring. The pituitary-gonadal axis is transiently activated in early infancy with peak serum levels of FSH, LH, inhibin B, and testosterone around 3 months of age (7, 8). This pituitary-gonadal activation can be used in the clinical evaluation of infants with suspected hypogonadism (9, 10). It is assumed that the levels of reproductive hormones during infancy are predictive of adult reproductive function (8). Therefore, we have studied serum levels of reproductive hormones in 3-month-old boys conceived by ART and in naturally conceived (NC) boys.

	Subjects and Methods

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Study participants and design

A total of 1182 boys were examined at approximately 3 months of age (3.1 months; 2.6–4.3 months) (median; 2.5th–97.5th percentiles). For pre- and post-mature children [gestational age (GA) < 37 and > 42 wk, respectively], the date of examination was corrected for the expected date of delivery (266 d after conception). A total of 249 boys were conceived by ICSI or IVF, and 933 were NC. A team of seven doctors performed all examinations, which were strictly standardized through workshops, in which two doctors examined the same child.

Boys conceived by ICSI or IVF

Between 1998 and 2005, all families who were treated at the Fertility Clinic, Rigshospitalet, were consecutively invited to participate. In this longitudinal prospective cohort study from which other aspects were published earlier (11), we achieved a participation rate of 55.2% and 32.8% for the ICSI and IVF groups, respectively. A total of 125 boys conceived by ICSI were included, and in 74 of these boys (59.2%), successful venipuncture resulted in a blood sample for hormone analyses. There were 124 boys conceived by IVF also included, and in 88 of these boys (71.0%), successful venipuncture resulted in a blood sample for hormone analyses. None of the children were conceived with donor semen. Data on paternal sperm parameters before preparation at the day of fertilization were collected from medical records. The indications for fertility treatment were divided into four groups: male factors (poor or very poor semen quality); female factors (tubal factor, myomas, endometriosis, anovulation); others (unexplained infertility or earlier fertilization failure); and combinations (Table 1). In subanalyses, 80 boys conceived by ICSI exclusively because of male factors after exclusion of seven boys conceived by semen washed for antibodies and 73 boys conceived by IVF exclusively because of female factors were included, of which 43 (53.8%) and 56 (76.7%), respectively, had a blood sample taken.

NC boys

This control group originated from a contemporary prospective birth cohort study investigating genital development (12). Blood samples were available from 622 infant boys (66.7% of the entire studied cohort of healthy children). Mothers were recruited consecutively in the first trimester of pregnancy, and the children were born between 1997 and 2001.

Anthropometric measurements

Birth data and obstetric history were obtained from medical records. At 3 months of age, weight was measured on a digital scale (Baby-Scale model; Solotop Oy, Helsinki, Finland) to the nearest 0.005 kg; the mean of 3 was calculated. The length of the children was measured in the supine position with a portable infantometer (Kidimeter; Raven Equipment Ltd., Essex, UK) to the nearest 0.1 cm; the mean of 3 was calculated. Penile length was measured as flaccid penile length without erection with a slide gauge (Baty Int., Burgess Hill, West Sussex, UK). Confidence intervals (95%) for intraobserver and interobserver variations were ±0.36 and ±0.67 cm, respectively (13).

Laboratory methods

Nonfasting peripheral venous blood samples were taken from an antecubital vein after local anesthesia between 0900 and 1600 h. One attempt was made in each child. Thus, blood samples could not be obtained from all infants due to an unsuccessful procedure or parental objection. Samples were separated by centrifugation and stored at –20 C until analysis. Three (4.2%) ICSI boys, two (2.3%) IVF boys, and eight (1.3%) NC boys had serum testosterone levels below the detection limit. There were 38 (55.1%) ICSI boys, 41 (49.4%) IVF boys, and 250 (42.2%) NC boys who had serum estradiol levels below the detection limit. For statistical analyses, blood sample results under the detection limit were assigned the detection limit.

Assays

Blood samples were analyzed consecutively over a 5-yr period. The technician was blinded to the type of conception.

Testosterone was measured by a solid-phase RIA (Diagnostic Products Corp., Los Angeles, CA). The detection limit was 0.23 nmol/liter. The intraassay and interassay coefficients of variation (CVs) were 7.6% at 6.7 nmol/liter and 9.7% at 8.0 nmol/liter, respectively. The same internal serum controls for testosterone (at three different concentration levels) were included in each assay during the study period, and no significant time trends were apparent. The normal median value for testosterone in fertile men aged 31 yr [25–42 (n = 318)] was 22.4 nmol/liter (10.8–36.4) (6). Free testosterone (pmol/liter) was calculated according to Vermeulen et al. (14). We applied an average albumin concentration for 3-month-old children of 40 g/liter (15) because albumin was not measured individually.

LH, FSH, and SHBG were measured by a two-sided time-resolved fluoroimmunometric assay (Delfia; Wallac, Inc., Turku, Finland). The detection limit for LH and FSH was 0.05 IU/liter and for SHBG 0.23 nmol/liter. The intraassay CV for LH was 3.0% at 0.3 U/liter, FSH 2.1% at 1.3 U/liter, and SHBG 5.1% at 96 nmol/liter. The interassay CV for LH was 4.4% at 1.4 U/liter, FSH 2.8% at 6.8 U/liter, and SHBG 4.0% at 101 nmol/liter.

Serum inhibin B was measured in a double antibody enzyme-immunometric assay using monoclonal antibody raised against the inhibin ß_B subunit in combination with a labeled antibody against the -subunit. The detection limit was 20 pg/ml. The intraassay and interassay CVs were 10.0% at 241 pg/ml and 8.6% at 233 pg/ml, respectively.

Total serum estradiol was measured by RIA (Pantex Corp.; Immunodiagnostic Systems Ltd., Santa Monica, CA). The detection limit was 18 pmol/liter. The intraassay and interassay CVs were 7.5% at 75 pmol/liter and 12.3% at 96 pmol/liter, respectively.

Statistics

Descriptive statistics are given as median hormone values and reference ranges (2.5th and 97.5th percentiles). Weight for GA was expressed as the deviation from the expected mean (16) and categorized into appropriate weight for gestational age (AGA) (birth weight between –2 and +2 SD) (17), small for gestational age (SGA) (<–2 SD), and large for GA (>+2 SD).

Differences in hormone concentration and penile length between types of conception were tested by Kruskal-Wallis tests, and thereafter compared two-by-two using the Mann-Whitney U test. Hormone levels in relation to levels in NC boys were calculated using: [value 1 (ICSI) – value 2 (NC)]/value 2 (NC)*100. Spearman’s correlation was used for associations between paternal and male offspring serum testosterone levels.

Prevalences were analyzed by the ² test for differences between various maternal and neonatal factors comparing ICSI or IVF with NC. If there were less than five observations, Fisher’s exact test was done. Binary logistic regression was used for differences between types of conception concerning gestational diabetes adjusting for maternal age and twinning.

Before multivariate analyses (general linear models) were performed, square root (serum testosterone and free serum testosterone) and logarithm transformations [serum LH/serum testosterone (natural logarithm) and paternal serum testosterone (log)] were applied to improve the approximation to a normal distribution. Depending on the analysis, the following confounders were included in the model: type of conception (NC as reference), twinning (singleton/twins), cryptorchidism (yes/no), hypospadias (yes/no), maturity (mature as reference), weight for GA (AGA as reference), parity (primiparous as reference), gestational diabetes (yes/no), maternal age at delivery, infant serum testosterone, physical age (number of days from conception to blood sampling), or postnatal age.

The statistical analyses were carried out using SPSS version 14 (SPSS, Inc., Chicago, IL).

Ethical aspects

The study was performed according to the Helsinki declaration II, and approved by the local Ethical Committee [(KF) 01–030/97; (KF) 01–090/98] and the Danish Registry Agency (1997-1200-074; 1998-1200-262). The parents gave informed written consent.

	Results

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Tables 1 and 2 show clinical characteristics of infants and their parents. Semen quality in the ICSI group was significantly lower compared with the IVF group (Table 1). Mothers of ICSI and IVF boys were significantly older compared with NC boys [33.4 yr (26.6–39.2), P > 0.001 and 34.4 yr (25.7–40.1), P > 0.001 compared with 30.6 yr (22.8–39.6), respectively]. Fathers of ICSI and IVF boys were significantly older compared with NC boys [36.5 yr (28.8–55.1), P > 0.001 and 35.1 yr (28.1–49.7), P > 0.001 compared with 32.0 yr (24.6–46.2), respectively]. Mothers of boys conceived by ICSI or IVF were more often primiparous, and had a higher frequency of gestational diabetes and caesarean section. They more often gave birth to twins, and premature and SGA children (Table 2). The prevalence in gestational diabetes did not differ between groups after adjusting for confounders. No significant differences were found in the prevalence of hypospadias or cryptorchidism between the groups (Table 2).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Serum testosterone in boys at 3 months of age conceived by ICSI because of male infertility factors and boys conceived by IVF because of female infertility factors compared with NC boys. ICSI compared with NC after adjusting for twinning, maturity, weight for GA, and physical age in the multivariate analysis (*). Data from boys with hypospadias and/or cryptorchidism were excluded.

Paternal serum testosterone levels in ICSI fathers (n = 73) were 12.1 nmol/liter (6.6–23.7). Paternal serum testosterone concentration correlated significantly to serum testosterone in its male offspring; paired father-son samples were available in 44 ICSI and 32 IVF (r = 0.232; P = 0.04). There was no significant correlation between the LH to testosterone ratio in fathers and sons.

In the whole study population, boys conceived by ICSI or IVF had a significantly shorter penile length (3.6 cm; 2.8–4.7; and 3.6 cm; 2.8–4.6, respectively) compared with NC boys (3.9; 3.1–4.8 cm; P < 0.001). However, after selecting boys conceived by ICSI exclusively because of male factors and boys conceived by IVF exclusively because of female factors, only IVF boys had significantly shorter penile length (ICSI: B = –0.082, P = 0.32; IVF: B = –0.209, P = 0.006).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In this large prospective cohort study, we found reduced serum testosterone levels and elevated LH to testosterone ratios in 3-month-old boys conceived by ICSI. Our findings may represent subtle Leydig cell dysfunction among the ICSI boys. ICSI was first introduced in the 1990s. Thus, no data are yet available on fecundity of adults conceived by ICSI. Three-month-old infants have a well-described "window of opportunity" to study their testicular function (7, 8, 9, 10, 18, 19), which is regarded to be predictive of reproductive capacity in adulthood (8).

We did not find any significant elevation of serum LH levels in ICSI boys, which might be due to the pulsatility of LH that was reported in male newborns, and if still present at 3 months of age, may have lowered the chance to detect potential LH differences (20).

ICSI is most often used when the infertility of a couple is caused by male factors, whereas IVF is predominantly used for female factors. Familial clustering of decreased testicular function is well described (2, 3, 4, 5), suggesting that genetic factors are important for male infertility. However, few cases of male infertility can currently be attributed to known genetic defects (21, 22, 23). A crucial question is whether or not decreased Leydig cell function also can be inherited from father to son. Importantly, a recent study showed that infertile men had on average 18% lower serum testosterone than fertile men (6), and we speculate that in our study, some of the boys may have inherited Leydig cell dysfunction from their fathers. Supporting our theory of vertical transmission of low serum testosterone levels, we found a significant correlation between paternal and infant serum testosterone.

Although the hormonal changes may be inherited from their fathers, they could also be due to hormonal treatment of the mothers or, alternatively, be an effect of the modern-assisted reproductive techniques themselves. This was illustrated by a recent study (24) that found a reduced sperm quality in young men whose mothers had received hormonal treatment to obtain pregnancy.

Our results may be influenced by the higher maternal age, prevalence of gestational diabetes, primiparous status, and number of twins for mothers of ICSI boys. Divergent studies are published on the effect of maternal age and parity on the risk of diabetes type 1 and cryptorchidism in childhood (25, 26, 27). Gestational diabetes is associated with an increased risk of infant malformations (28, 29). Twinning is also known to be associated with an increased risk of malformations and prematurity (30). However, inclusion of all possible confounders in our analyses did not change our results.

The pituitary-gonadal axis is transiently activated around the age of 3 months (7, 8, 18, 19), however, the exact timing of the hormonal peak is unknown and likely to vary between individuals. We examined boys only once at 3 months of age, which was corrected for the expected date of delivery. Including the age of examination in the analyses did not alter our findings.

Impaired fetal growth may influence postnatal testicular function (31, 32). Because boys conceived by ICSI were more likely to be born SGA (8.9%) compared with NC boys (3.4%), our findings of lower testosterone could theoretically be caused by restricted intrauterine growth. However, selecting only boys with normal birth weight and normal GA at birth for statistical analyses did not alter our findings. The fetal programming theory (33) suggests that an adverse intrauterine environment may cause a permanent alteration of some metabolic and endocrine set points. Reassuringly, we did not find any differences in the FSH-inhibin B axis, suggesting normal Sertoli cell function in ART boys at 3 months of age.

Endogenous testosterone contributes to postnatal phallic growth (13). We found significantly but only slightly smaller penile length in ART boys. The significance of this is yet unknown, and only IVF boys had a significantly shorter penile length in the multivariate analysis. It would have been more supportive of our hypothesis of inherited poor Leydig cell function if differences in penile lengths between the groups paralleled the hormonal differences. A large register-based study (34) demonstrated a significantly higher prevalence of hypospadias among ICSI boys compared with NC boys. This is in line with the relatively high prevalence of hypospadias (1.6%) in ICSI boys compared with NC boys (1.2%), although this difference was not statistically significant.

In conclusion, we found a significant reduction (27%) in serum testosterone levels and a significantly higher LH to testosterone ratio in boys conceived by ICSI compared with NC boys. This points toward a subtle impairment of Leydig cell function in boys conceived by ICSI using sperm from oligozoospermic men. The clinical relevance of our findings remains to be clarified. However, our findings should raise concern because ICSI is increasingly used to overcome male infertility.

	Acknowledgments

 
We thank all participating families, especially the children. The Nordic Cryptorchidism Study Group was involved in examination of the children. We appreciate the skilled help of nurse Helle Kelkeland, students, and the technicians of the University Department for Growth and Reproduction. We also thank secretary Bente Wedel Larsen and the staff of the Fertility Clinic for practical help.

	Footnotes

 
This work was supported by The University of Copenhagen, The Danish Medical Research Council (no. 9700833 and 9700909), The European Commission (QLK4-CT1999-01422, QLK4-2001-00269), Turku University Central Hospital, the Academy of Finland, and the Novo Nordisk Foundation.

C.M.K., A.N.A., A.L., N.E.S., and A.J. have nothing to declare. K.M.M. received lecture fees from Novo Nordisk, Pfizer, and Ferring.

First Published Online April 24, 2007

Abbreviations: AGA, Appropriate weight for gestational age; ART, assisted reproduction technologies; CV, coefficient of variation; GA, gestational age; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; NC, naturally conceived; SGA, small for gestational age.

Received January 16, 2007.

Accepted April 13, 2007.

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