Successful IVF pregnancy despite inadequate ovarian steroidogenesis due to congenital lipoid adrenal hyperplasia (CLAH): a case report

Abstract

Steroidogenic acute regulatory protein (StAR) mutations are the most frequent aetiologies of congenital lipoid adrenal hyperplasia (CLAH). Phenotypes may vary, and puberty may be absent in affected individuals. To date, only two pregnancies have been described in 46,XX CLAH patients with StAR mutations; these patients exhibited ovarian steroidogenesis along with spontaneous puberty and menarche and normal menses. The patient described here presented with CLAH caused by the homozygous (unreported, 1 bp) deletion c.719del in the StAR gene, which was diagnosed after acute adrenal insufficiency when the patient was 10 days old. The patient did not undergo spontaneous puberty, so puberty was induced by HRT when the patient was 13 years old. At the age of 25 years, the patient was referred to our reproductive unit because she desired to conceive. An initial cycle of clomiphene, stimulation produced follicular growth with two mature follicles measuring 18 and 15 mm, respectively, but the plasma oestradiol levels remained low (18 pg/ml) and the endometrium was thin (3 mm). Pregnancy was finally achieved after ovarian stimulation, IVF and transfer of frozen–thawed embryos after endometrial preparation with HRT. A normal female child was delivered following a 40 weeks’ uneventful pregnancy. We therefore report the first IVF pregnancy achieved in a 46,XX CLAH patient homozygous for a StAR mutation, with inadequate ovarian steroidogenesis and no spontaneous puberty.

StAR gene, congenital lipoid adrenal hyperplasia, adrenal failure, fertility, pregnancy

Introduction

Congenital lipoid adrenal hyperplasia (CLAH) is a severe genetic disorder of steroidogenesis in which there is a complete or nearly complete absence of all circulating steroids (Kim, 2014). Steroidogenic acute regulatory (StAR) gene mutations are the most frequent aetiology of CLAH, but to our knowledge, <100 cases have been reported in the literature. These mutations span the entire StAR gene, which is localized in 8p11.2 (Bhangoo et al., 2006). The StAR protein is involved in cholesterol transport from the outer to the inner mitochondrial membrane, where cholesterol is used as a substrate for the first step of steroidogenesis (Lin et al., 1995). In 46,XX patients with a StAR mutation, a wide clinical spectrum of disorders is observed, from no puberty at all (Nakae et al., 1997) to normal puberty with regular menses (Bose et al., 1997).

Thus far, pregnancy has been reported in only two women with a StAR gene mutation who presented with spontaneous puberty and menarche; these pregnancies were facilitated using two different types of intervention (clomiphene citrate treatment and IVF) (Khoury et al., 2009; Sertedaki et al., 2009).

Herein, we report the unusual case of a 46,XX CLAH patient, homozygous for a 1 bp deletion in the StAR gene, namely, c.719del, and with neonatal adrenal insufficiency and no spontaneous puberty, in whom an IVF pregnancy was attained despite a lack of StAR-dependant ovarian steroidogenesis.

Case report

Patient history

The patient provided consent for the publication of this report. She was born at term of consanguineous parents of North African origin after a normal pregnancy and delivery. At birth, she was healthy and of normal size (weight: 3.570 kg, height: 52 cm) and had external female genitalia. Her family history included a previous 46,XY child who was phenotypically female and died at 4 years of age; this death was related to acute adrenal insufficiency associated with sepsis. During the neonatal assessment of our patient, a diagnosis of acute adrenal insufficiency was made after 10 days of life. This adrenal insufficiency manifested as fever, growth and weight failure, vomiting, dehydration, hyponatraemia, hyperkalaemia associated with back and neck hyperpigmentation, low levels of all adrenal steroids (cortisol 1.9 µg/100 ml, dehydroepiandrosterone sulphate <15 ng/ml, undetectable progesterone and 17-OH progesterone) and elevated adrenocorticotrophic hormone and renin plasmatic activity levels (>1100 pg/ml and >35 ng/ml, respectively). The combined deficiencies of mineralocorticoids and glucocorticoids suggested a defect in the first steps of steroidogenesis and CLAH in this 46,XX patient.

Mineralocorticoid- and glucocorticoid- replacement therapies were initiated and produced an immediate clinical response; intravenous (IV) glucocorticoids were used during the acute crisis and then hydrocortisone (50 mg/m²/day), fludrocortisone (25 µg 3 times per day) plus salt substitution (15 mmol/kg/day).

Sonography showed normal kidneys, adrenals and internal female genitalia. The karyotype of the patient was 46,XX. Under hormonal substitution, the growth and neurological development of the patient were normal until she reached the age of puberty. At 12 years old, the patient exhibited no clinical pubertal development or pubic or axillary hair. A pelvic ultrasound revealed the presence of an infantile uterus (26 mm) and ovaries with antral follicles (but hormonal data are not available). At the age of 13 years and 6 months, the patient began HRT to induce puberty; this therapy consisted of oestradiol alone (200 µg, increased to 500 µg for 9 months) and then oestradiol in association with progesterone (chlormadinone at 5 mg and then 10 mg for 21 months), followed by triphasic therapy with oestradiol and progesterone (norethisterone acetate). The mineralocorticoid- and glucocorticoid therapies at this time consisted of hydrocortisone 30 mg, 3 times per day and fludrocortisone 60 µg, 3 times per day.

Genetic analysis

Molecular analysis was performed when the patient was 9 years old (Portrat-Doyen et al., 1996) after obtaining informed consent. Genomic DNA was extracted, and selective amplification of all exons and the exon–intron boundaries of the StAR gene was performed by PCR, after which the gene was sequenced (Bose et al., 1996). Sequence variants were designated according to the Human Genome Variation Society recommendations (www.hgvs.org/rec.html) using the NCBI reference sequences NC_000008.10, NM_000349.2 and NP_000340.2 built on GRCh37/hg19.

Molecular analysis of the StAR gene revealed a homozygous frameshift deletion, NM_000349.2, namely, c.719del, in exon 6, which led to a protein of 320 (instead of 286) amino acids in length: p.Thr240Serfs*81. This mutated protein did not contain the last 46 carboxy-terminal residues of StAR and was predicted to have no StAR activity (Lin et al., 1995; Arakane et al., 1996). As expected in autosomic recessive transmission, both of the patient's parents were heterozygous for the mutation, as was one of the patient's sisters.

Fertility management

The patient was referred to our reproductive medicine unit at the age of 25 because she desired a pregnancy. She presented with facio-troncular obesity (weight: 82 kg, height: 158.5 cm, BMI: 32.6 kg/m²). The patient also presented with Tanner scores of four for the breasts and two and three for axillary and pubic hair, respectively, and was given HRT composed of oestradiol and progesterone (norethisterone acetate).

Failure of ovarian stimulation with clomiphene citrate

The sperm parameters of the patient's husband were normal. We first prescribed clomiphene stimulation (50 mg per day for 5 days) following a report by Khoury et al. (2009), describing a pregnancy occuring after clomiphene stimulation in a CLAH patient. In our particular patient, we observed the growth of two follicles (18 and 15 mm), but plasma oestradiol levels remained low (13–28 pg/ml from the 17th to the 26th day of stimulation, respectively) with luteinizing hormone at 12 IU/l and a thin endometrium (3 mm). Ovulation was triggered using choriogonadotropin alfa, and the luteal phase was supported with vaginal progesterone. However, no pregnancy was achieved. In the absence of ovarian steroidogenesis and due to the risk of premature ovarian failure because of CLAH, we proposed IVF management.

Ovarian stimulation for IVF

Before the first attempt at IVF, the antral follicle count was 11 and the AMH level was 2.11 ng/ml (Beckman Coulter, Brea, CA, USA). In September 2014, we performed a long agonist protocol with down-regulation using long-acting triptorelin and ovarian stimulation with 300 units per day of menotropin. We prescribed concomitant HRT with oral oestradiol (2 mg b.i.d.) to induce endometrial proliferation for fresh embryo transfer. Triggering using 10 000 units of chorionic gonadotropin was performed on Day 14 of stimulation, resulting in four follicles measuring 20 mm and four follicles measuring 14–15 mm. Moreover, the endometrial thickness measured 11 mm. Vaginal micronized progesterone was introduced the day after ovulation was triggered (600 mg per day). We then retrieved eight oocytes and obtained six top-quality diploid embryos. We transferred two fresh embryos and froze four 2-day-old embryos. However, pregnancy was not achieved.

Pregnancy after replacement of a frozen embryo and HRT

In November 2014, artificial endometrial preparation was performed, for the transfer of a frozen–thawed embryo, starting with daily administration of 4 mg of oral oestradiol. After an ultrasound determined that the endometrial thickness was 9.5 mm, vaginal progesterone was administered (600 mg daily) and two embryos were transferred 3 days later. Spontaneous abortion of a singleton gestation occurred at 8 weeks of gestation. A second transfer of two frozen–thawed embryos was performed in February 2015 after the same hormonal endometrial preparation, and a pregnancy was attained. HRT was maintained during the first trimester of pregnancy. The pregnancy proceeded uneventfully, with an appropriate substitution of glucocorticoids: hydrocortisone, 10–30 mg 3 times per day, depending on clinical symptoms (there were two episodes of vomiting during the first trimester that required brief hospitalization, hydration and IV glucocorticoids) and fludrocortisone, 50 µg 2 times per day. In November 2015, a normal female child was delivered after 40 weeks of gestation, weighing 3.150 kg. The patient received 50 mg of IV glucocorticoids during the delivery. No perinatal problems were observed, and the usual substitution treatment (hydrocortisone 10 mg 3 times per day and fludrocortisone 50 µg 2 times per day) was prescribed to the patient during the post-partum period.

Discussion

To our knowledge, this is the first report of a full-term pregnancy in a CLAH patient with a homozygous mutation of the STAR gene that led to an absence of spontaneous puberty and menarche. In the literature, one 46,XX Japanese patient who was heterozygous for p.Q258X and A218 V mutations and who had no pubertal development until she was 16.4 years old has been described (Nakae et al., 1997). Other described patients underwent spontaneous breast development that was delayed compared to that in unaffected girls. Papadimitriou et al. (2006) suggested that in patients with CLAH, ovarian insufficiency begins in infancy. Moreover, the capacity of the ovary to maintain steroid synthesis until or after puberty confirms the ‘two-hit model’ suggested by Bose et al. (1996). Indeed, the first step in this model involves the initial loss of StAR-dependent steroidogenesis caused by genetic mutation. Thereafter, cellular damage due to the accumulation of cholesterol esters leads to loss of the already low-level StAR-independent steroidogenesis. This theory explains why 46,XX females can undergo spontaneous breast development and sometimes cyclic vaginal bleeding (Bose et al., 1997; Nakae et al., 1997), i.e. because of the absence of steroidogenesis in foetal cells and weak tropic stimulation by gonadotropins until the age of puberty in girls. However, accumulating lipids in follicular cells inhibit progesterone synthesis; thus cycles are anovulatory and can produce large cysts, which in turn can lead to ovarian torsion (Shima et al., 2000, Jin et al., 2011). A low dose of HRT can effectively prevent ovarian cyst formation (Sertedaki et al., 2013).

In the first pregnancy in a CLAH patient, described in Sertedaki et al. (2009), the patient had an 11-bp deletion in the StAR gene and presented with spontaneous puberty and menarche and normal menses at 24 years of age (Sertedaki et al., 2009). The authors reported that during the first attempt at IVF, endogenous oestrogen secretion was detected, although the level was low after 11 days of ovarian stimulation (115 pg/ml). Additionally, Khoury et al. (2009) described a patient, with an L275P StAR mutation, who presented with spontaneous puberty and menarche. Ovulation was reported after treatment with clomiphene citrate, indicating a functional pituitary–ovarian axis and effective ovarian steroid synthesis (Khoury et al., 2009). Our patient did not present with spontaneous puberty, and despite the presence of follicular growth after ovarian stimulation with clomiphene citrate, the plasma oestradiol levels remained very low.

As previously reported by Gougeon, oestrogen may not play a key role in folliculogenesis (Gougeon, 1996), but in our case, chronic exogenous oestrogen treatment made it difficult to verify this conclusion.

Acknowledgements

We are grateful to Yves Morel for his corrections and advice on genetics and to Gilbert Simonin for the paediatric follow-up of this patient.

Authors’ roles

B.C. supervised the study and followed the patient during fertility management. F.A. wrote the manuscript and adapted the substitution treatment. J.P., M.J., F.R.B., R.R. and T.B. contributed to the acquisition of data and the follow-up of the patient. All authors were involved in writing the manuscript and have approved the final version.

Funding

No funding was received for this study.

Conflict of interest

None declared.